Chloroplasts engineered to express pharmaceutical proteins

ABSTRACT

Vaccines for conferring immunity in mammals to infective pathogens are provided, as well as vectors and methods for plastid transformation of plants to produce protective antigens and vaccines for oral delivery. The vaccines are operative by parenteral administration as well. The invention also extends to the transformed plants, plant parts, and seeds and progeny thereof. The invention is applicable to monocot and dicot plants.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/915,666 filedNov. 27, 2007, which claims priority to PCT/US06/21024 filed May 30,2006, which claims the benefit of U.S. Ser. No. 60/685,734, filed May27, 2005, which are incorporated herein in their entirety by reference.

BACKGROUND

Progress has been made in engineering plant cells to produce usefulproteins. For example, plants have been shown to express potentiallymedically important proteins that may be used for immunization againstpathogens. Many infectious diseases require booster vaccinations ormultiple antigens to induce and maintain protective immunity. Advantagesof plant-derived vaccines include the delivery of multiple antigens, lowcost of production, storage & transportation, elimination of medicalpersonnel and sterile injections, heat stability, antigen protectionthrough bioencapsulation, the generation of systemic & mucosal immunityand improved safety via the use of a subunit vaccine and absence ofhuman pathogens. Despite cases of successful expression of proteins, thedevelopment of plant derived medically important compositions is stillin its formative stages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: CTB-Pris Construct and Site of Integration into the ChloroplastGenome: Insertion of 5′UTR-CTB-human proinsulin into the chloroplasttransformation vector pLD and the site of integration into thechloroplast genome between the trnI and trnA genes.

FIG. 2: Western blot analysis of chloroplast transgenic lines probedwith proinsulin antibody: Lane 1 E. coli crude extract expressingCTB-Pins, lane 2 untransformed plant extract, lanes 3-5 plant extract oftransgenic lines.

FIG. 3A: Southern blot probed with BamHI/BglII 0.81 kb flankingsequence. Gene specific probe (0.36 kb) was obtained by MfeI/NotIdigestion of pLD-5CP vector. 3B: Illustration of untransformed andtransformed chloroplast genomes at the site of integration oftransgenes. Untransformed & transformed plant DNA was digested withAflIII and AflIII. The expected size for each fragment is shown alongwith the hybridization site for the flanking sequence probe and genespecific probe. 3C: Southern Blot with gene specific probe: Lanes 1-5:DNA from transgenic lines; lane 6: untransformed wild type. 3D: SouthernBlot with flanking sequence probe: Lane 1: untransformed wild type,lanes 2-6: transgenic lines.

FIG. 4: A) Haematoxylin & Eosin staining of a section of the pancreas(showing an islet: isl) of a mouse treated with CTB-Pins for 7 weeks.There is no cellular infiltration inside the islet. Lymphocytes areshown outside the islet (arrow in A). In 4 B) arrows indicate theborders of an islet in the pancreas of a mouse treated with CTB-GFP (acontrol group). Blue dots show cellular infiltration of the islet. FIG.4C shows a big islet with severe lymphocytic infiltration in a mousetreated with untransformed (UN-Tr) plant leaf material. In 4 D) a severelymphocytic infiltration in a mouse treated with interferon-GFP(IFN-GFP) is shown.

FIG. 5: Scoring (S) the insulitis according to the severity of thelymphocytic infiltration of the pancreas Langerhans islets. Score 1indicates no or pre-islet infiltration, minimal infiltrations werescored 2, moderate infiltrations were scored 3 and severe infiltrationswere scored 4. When more than 80% of the islets were infiltrated, thescore was 5.

FIG. 6: Lymphocytic infiltrations (insulitis) were scored by blindlyevaluating 50 sections per pancreas of each animal in differentexperimental groups as indicated. The NOD mice treated with CTB-Pinsscored significantly lower (P<0.05) than the untransformed (UN-Tr)plant, interferon-GFP (IFN-GFP), or CTB-GFP plant treated groups. ANOVAwas done through Excel, and the P value is less that 0.001. The barsrepresent the standard deviation.

FIG. 7: Insulin immunoreactivity in Langerhans islets of a mouse treatedwith CTBPins (A). In 7 B, Caspase-3 immunostaining in the same sectionis shown in the red channel. Merged picture of A and B is shown in 7C.7D) A view of the pancreas which shows the remnant of a large langerhansislet of the mouse treated with untransformed plant leaf material. E)shows the Caspase-3 immunoreactivity in the same section taken in redchannel. F) shows the merged picture of D and E; here, all the remainingcells which are also depleted of insulin, are expressing the activecaspase-3.

FIG. 8: Interleukin 10 (IL10) immunoreactivity in the pancreas of threemice treated with untransformed plant leaf material A, B, and C. Bloodvessels (BV) and the langerhans islets (isl) are indicated. Nosignificant IL10 immunostaining can bee seen in or around the islets oraround the blood vessels. D, E, and F show the islets of mice treatedwith CTB-Proinsulin. Small arrows indicate perivascular infiltration ofIL10 expressing lymphocytes. Large arrows indicate IL10 positivelymphocytes inside or around the islets.

FIG. 9: Interleukin-4 (IL4) immunoreactivity in the pancreas of micetreated with IFNGFP or CTB-GFP or CTB-Pins plant leaf material. Bloodvessels (BV) are free of perivascular lymphocytic infiltration and nosignificant IL4 positive cells can be seen around the islets (arrowsdemarcate the islets). A large number of IL4 positive cells are shownaround the islets of CTB-Pins treated NOD mice.

FIG. 10: Serum levels of IgG1 in NOD mice treated with CTB-Pinsexpressing plant leaf material as compared to the control groups treatedwith untransformed plant, the CTB-IFN or CTB-GFP plant expressing leafmaterial.

FIG. 11: PCR analysis of Wild type and putative transformants ofpLD-5′UTR-His-CTB-NSP4. A: PCR using specific primers land within thenative chloroplast genome (3P/3M) to yield a 1.65 kb product and 5P/2Mprimers to yield 2.5 kb product. B: Lane 1: 1 kb plus DNA ladder, Lane2:Negative control (Wild type) Lane 3-6: Transgenic lines of HisCTB-NSP4,Lane 7: Empty, Lane 8: Positive control (Interferon clone). C: Lane 1: 1kb plus DNA ladder, Lane 2: Negative control (wild type), Lanes 3-6:Transgenic lines of HisCTB-NSP4, Lane 7: Empty, Lane 8: Positive controlpLD 5′UTR-HisCTB-NSP4 plasmid.

FIG. 12 Southern Blot analysis of CTB-NSP4 T0 plants. Schematic diagramof the products obtained from digestions of A: Wild type untransformedplants show a DNA fragment of 5 kb. B: Two DNA fragments of 4.3 kb and 2kb indicate plants that are transformed with pLD-5′UTR-HisCTB-NSP4. C: ADNA fragment of 11 Kb is seen for transgenic lines with gene specificprobe D: Southern with flanking sequence probe of CTB-NSP4 transgenicplants showing homoplasmy. Lane 1: 1 kb plus DNA ladder, Lane 2: Wildtype, Lanes 3-8: CTB-NSP4 transgenic lines E: CTB-NSP4 gene specificprobe showing the presence of CTB-NSP4 gene in the transgenic plants.Lane 1: 1 kb plus DNA ladder, Lane 2: Wild type, Lanes 3-6: CTB-NSP4transgenic lines

FIG. 13: Immunoblot analysis of crude plant extracts expressingCTB-NSP4. Lane 1: Molecular weight markers, Lane 2-3: Boiled T0transgenic plant samples, Lane 4-5: Unboiled T0 transgenic plant samples(20 ug of crude plant extract was loaded). Lane 6: Wild type, Lanes 7:Empty, Lane 8: bacterial CTB-NSP4₉₀ fusion protein purified from EcoliBL 21 cells (0.9 ug).

FIG. 14. Quantification of CTB-NSP4 fusion protein expression levels intransgenic plants (T0 generation). Expression levels in % total solubleprotein (TSP) of CTB-NSP4 expressed in Young, Mature and Old leavesunder continuous light illumination observed for 0 to 5 days. TheCTB-NSP4 expression levels reached a maximum of 2.45% of TSP in matureleaves by day 1 under continuous light and the expression levelsdeclined to 0.6% of TSP by Day 5.

FIG. 15: Schematic steps to clone pLD-AB-NS3

(a) Amplification of 5′ terminal 134 bp of NS3 gene using PCR. SacI andSnaBI and NotI are introduced for further subcloning. (b) Cloning of PCRproduct in p-Bluescript between Sad and NotI. (c) pcDNA3.1-NS3 vectordigested with BstXI and EcoRV and cloned between same sites inp-Bluescript. NS3 gene cloned in p-Bluescript between Sad and EcoRV. (d)NS3 gene in p-Bluescript digested with SnaBI and HindIII and cloned inpCR2.1 between the same sites and upstream of 5′UTR. (e) NS3 gene and5′UTR (2.1 kb) digested from pCR2.1 with EcoRI and EcoRV and cloned inbetween same sites in pLD-AB-Ct vector.

FIG. 16: Nicotiana tabacum chloroplast genome

The pLD contains the chloroplast transfer RNAs coding for Isoleucine andAlanine (trnI and trnA). These homologous flanking DNA sequences directthe insertion of the Prrn/aadA/5′UTR/NS3′UTR genes into the chloroplastgenome by two homologous recombination events.

FIG. 17: Chemiluminescent Detection of E. coli-expressed NS3

Total E. coli proteins were separated on SDS-PAGE and detected withmonoclonal anti-NS3 as the primary antibody. The secondary antibody wasgoat anti-mouse IgG conjugated to horseradish peroxidase. Samples:Protein marker (lane1); Extracts of untransformed E. coli cells (lane 2and 3); Protein extracts from lysates of E. coli transformed withpLD-AB-NS3 (lane5 and spillover in lane 4).

FIG. 18: First Round of Selection

A. Shoots from bombardment of Petit Havana leaves appeared within 4weeks

B. Shoots from bombardment of LAMD-609 leaves appeared within 7 weeks

FIG. 19: Second Round of Selection

A. Pettit Havana shoots from first selection on 500 μg/ml spectinomycin

B. LAMD-609 shoots from first selection on 350 μg/ml spectinomycin

FIG. 20: Propagation of Petit Havana Transgenic Lines

A. Petit Havana transgenic lines in jars containing MSO 500 ug/mlspectinomycin.

B. Petit Havana transgenic plant in pots with no added antibiotic.

FIG. 21: 3P/3M PCR Analysis of Putative Petit Havana and LAMD TransgenicLines

A. 3P/3M primers annealing to sequences in the chloroplast genome ofPetit Havana and LAMD.

B. A 1.65 kb PCR product with 3P/3M primers: 1 kb DNA ladder (lane1);untransformed (−) petit Havana (lane 2); transgenic PH lines (lanes3-8); untransformed LAMD (lane 9, control); LAMD transgenic line (lane10).

FIG. 22: 5P/2M PCR of Putative Petit Havana and LAMD Transgenic Lines

A. 5P/2M primers annealing to sequences in the chloroplast genome ofPetit Havana and LAMD.

B. 0.8% agarose gel shows 3.7 kb PCR product utilizing 5P/2M primers; 1kb DNA ladder (lane1); 1 μg of pLD-AB-NS3 as the positive control (lane2); untransformed (−) Petit Havana (lane 3); untransformed (−)LAMD (lane4); transgenic petite havana lines (lanes 5-9); transgenic LAMD lines(lane10).

FIG. 23: Southern Blot using Flanking Probe

Confirmation of Chloroplast Integration and Determination ofHomoplasmy/Heteroplasmy in T₀ Generation.

A 810 bp probe containing chloroplast flanking sequences and DNAfragments of 4.47 kb indicate untransformed chloroplast. B. DNAfragments of 5.2 and 2.7 kb indicate transformed chloroplasts oftransgenic plants (lanes 1-8) and DNA fragments of 4.47 kb indicateuntransformed chloroplasts of transgenic plants (lane 9).

FIG. 24: Southern Blot using NS3 gene specific probe

A 2.1 kb NS3 gene specific probe was used. All transformed plants (lanes2-9) show 2.7 kb DNA fragment and the untransformed plant (lane1) doesnot show any DNA fragment.

FIG. 25: Western Blot of Transgenic plants expressing NS3

Plant tissue extracts separated on 10% SDS-PAGE with NS3 detected bymouse monoclonal antibody against NS3. Protein Marker (lane1);untransformed plant (lane 2); Blank-Sample buffer (lane 3); transgenicPH plant (lane 4); Mutant PH plant not expressing NS3 (lane 5);transgenic PH plant (lane 6); ransgenic LAMD plant (lane 7).

FIG. 26: Quantification of NS3 in Transgenic chloroplasts

A. Protein quantification by ELISA in young, mature and old transgenicleaves of LAMD of plant in 16 h light and 8 h dark (day 0), 1, 3 and 5day continuous illumination.

FIG. 27: Maternal inheritance

Seeds were sterilized and grown in MSO plates with spectinomycin (500ug/ul).

FIG. 28. pLD-CtV: Universal Chloroplast Expression Vector. The pLD-CtVcontains the 16S rRNA promoter, the aadA gene encoding spectinomycinresistance (selectable marker), and psbA 5′ & 3′ untranslated region toenhance translation in the light. The trnI and trnA inverted repeatregions allow for direct insertion of transgenes into the chloroplastgenome by two homologous recombination events.

FIG. 29. pLD-smGFP-IFNα5 (pLD-BB1) construct The pLD-smGFP-IFNα5contains the chloroplast transfer RNAs coding for Isoleucine and Alanine(trnI and trnA). These homologous flanking DNA sequences direct theinsertion of the aadA gene, 5′UTR/smGFP/furin/INFα5 cassette andregulatory sequences into the chloroplast genome by two homologousrecombination events.

FIG. 30. Expression of smGFP-IFNα5 in E. coli and Immunoblot AnalysisLanes: M: Markers, 1: Negative control (untransformed), 2: Positivecontrol (IFNα2b), 3: Positive control (IFNα5), 4-8: smGFP-IFNα5

FIG. 31. Selection and Regeneration: a. Primary Selection for TransgenicLines on Antibiotic Media. Shoots from bombardment of wild type DarkFire leaves. b. Secondary Selection for Transgenic Lines on AntibioticMedia. Shoots from Dark Fire on spectinomycin. c. Propagation 1.Transgenic lines in jar containing MSO with spectinomycin. 2. Transgeniclines and an untransformed plant in pots with no added antibiotic.

FIG. 32. 5P/2M PCR Analysis Both primers land on flanking sequences ofthe integrated construct. Integration into the chloroplast genomecreates a 3.0 kb PCR product. Untransformed tobacco plants should notproduce a PCR product. Lane 1: MW; 2-4: pLD-BB1 transformed plants A, B,F; 5: positive control; 6: untransformed plant (negative control).

FIG. 33. 3P/3M PCR Analysis The 3p primer lands on flanking region inthe native chloroplast genome and the 3m primer lands within the aadAgene. Integration into the chloroplast genome creates a 1.65 kb PCRproduct. Untransformed tobacco plants should not produce a PCR product.Lane 1: MW; 2-4: pLD-BB1 transformed plants A, B, F; 5: untransformedplant (negative control); 6: positive control; 7: MW.

FIG. 34. Southern Blot Analysis: Flanking Probe Southern BlotConfirmation of chloroplast integration and determination ofhomoplasmy/heteroplasmy in T_(o) generation of tobacco. The 0.81 kbprobe containing chloroplast flanking sequences generated an 8.3 kbfragment in the untransformed chloroplast genomes. In the transformedgenomes containing IFNα5 (A), 7.0 kb and 3.8 kb fragments weregenerated. In the transformed genomes containing the fusion ofsmGFP-IFNα5 (B,C,D), 6.3 kb and 3.8 kb fragments were generated whichshowed integration as well as homoplasmy.

FIG. 35. Southern Blot Analysis: IFNα5 probe Southern Blot using the 0.5kb IFNα5 probe. Transformed plant samples B,C, & D show the correctfragment size of 2.2 kb for the IFNα5 gene specific probe. No fragmentsare evident in the untransformed plant (WT). The IFNα5 probe was used asa positive control.

FIG. 36. smGFP Expression Viewed Under UV Light 1) Wild Type (a) &smGFP-IFNα5 (b) plants under normal light 2) Wild Type (a) & smGFP-IFNα5(b) plants under UV light 3)smGFP-IFNα5 plant under UV light 4) WildType plant under UV light FIG. 37. Immunoblot Analysis of Plants Westernblot analysis of protein expression of pLD-smGFP-IFNα5 in tobacco plants(Dark Fire cultivar): Lanes: M: Markers, 1: Positive control (E. colipLD-IFNα2b), 2: Positive control (E. coli pLD-IFNα5), 3: PositiveControl (E. coli pLD-smGFP-IFNα5), 4&6: skipped lanes, 5&7: Plantsamples B&C (pLD-smGFP-IFNα5), 8: Negative Control (D.F. Wild Type).

FIG. 38. ELISA Quantification of IFNα5 in transgenic chloroplasts:Protein quantification by ELISA in transgenic tobacco leaves. The amountof expression increased from 0.001% in the transformed plants containingIFNα5 to about 5% in the transformed plants expressing the fusionprotein, smGFP-IFNα5.

FIG. 39. PCR analysis for the confirmation of transgene integration. A)Schematic representation of the transgene cassette. B) 5P/2M—Theseprimers land on the aadA and trnA regions (flanking the CTB-GFP). A 2.9kb PCR product was obtained from the PCR analysis of transgenic plants.C) 3P/3M—The 3P primer lands on the native chloroplast genome and the 3Mprimer lands on the aadA gene. A 1.6 kb PCR product was obtained fromthe PCR analysis of the transgenic plants. Lane 1: 1 kb plus ladder.Lanes 2-5: Transgenic lines of CTBGFP. Lane 6: Positive Control. Lane 7:Empty. Lane 8: Wild-type Plant. D) Southern blot analysis of the plants.Lane 1: WT showing 4.4 kb fragment. Lanes 2-5: Transgenic plants showing4.9 and 2.2 kb hybridizing fragments. Flanking sequence shown in FIG.28A was used as the probe.

FIG. 40. Visualization of GFP fluorescence in transgenic plants under UVlight. A) Wild-type (untransformed) plant seen under UV light. B)CTB-GFP expressing leaf showing fluorescence observed under UV light. C)Wild-type leaf under a low magnification microscope. D) CTB-GFPexpressing leaf showing fluorescence under a low-magnificationmicroscope.

FIG. 41. Immunoblot analysis, furin cleavage assay and quantification ofCTB-GFP expressed in chloroplasts of transgeniclines. A) Immunoblotdemonstrating the expression of CTB-GFP in transgenic plant crudeextracts: Lane 1: Unboiledcrude extract of transgenic line A. Lane 3:boiled crude extract of transgenic line A. Lane 5: Unboiled crudeextract of transgenic line B. Lane 6: Boiled crude extract of transgenicline A. Lane 8: Purified CTB standard 200 ng. Lane 9: Wild-type plantcrude extract. Lanes 2, 4, 7: empty. B) Furin cleavage assay of theplant extract: Lane 1: Marker. Lane 2: CTB-GFP, pH 6.0, with furin, noPMSF. Lane 3: CTB-GFP no incubation, no furin. Lane 4: CTB-GFP pH 6.0with furin and PMSF. Lane 5: CTB-GFP, pH 7.0, with furin and PMSF. Lane6: CTB-GFP, pH 6.0, with PMSF, no furin. Lane 7: CTB-GFP, pH 6.0, noPMSF, no furin. Lane 8: Blank. Lane 9: Purified recombinant GFPstandard. C) Expression levels in % of CTB-GFP in total soluble protein(TSP) of the CTB-GFP expressing plants. D) GM1 ganglioside binding assayshowing the presence of CTB-GFP functional pentamers.

FIG. 42. Cryosections of the intestine and liver of the mice fed withCTB-GFP or wild-type plant leaves material. A) GFP in the ileum of amouse following oral delivery of the CTB-GFP expressing plant leafmaterial. Arrows show numerous columnar cells of the intestinal mucousmembrane, which have up-taken the CTB-GFP. Various cells in theconnective tissue beneath the epithelium also show the presence of GFP.B) Section of the ileum of a mouse fed by the wild-type (untransformed)plant leaf material. C) Section of the ileum of a mouse fed by theIFN-GFP leaf material. D) GFP in hepatocytes of a mouse liver followingoral delivery of CTBGFP expressing plant. E) Section of the liver of amouse fed by the wild-type plant material. F) Section of the liver of amouse fed by IFN-GFP expressing plant material. G) GFP in the spleen ofa mouse following oral delivery of CTB-GFP expressing plant. Arrows showvarious splenic cells with GFP. H) Section of the spleen of a mouse fedby the wild-type plant material. I) Section of the spleen of a mouse fedby IFNGFP expressing plant material. Scale bar: 50 μm.

FIG. 43. Immunohistochemical localization of the GFP in mouse ileum,liver, and spleen. A-C) are sections of the ileum of the mice fed withCTB-GFP expressing plant leaf. Arrows indicate presence of GFP in theintestinal epithelium as well as cells of the crypts. D) Shows a sectionof the ileum of a mouse fed with wildtype (untransformed) plant leafmaterials. E) GFP-immunoreactivity in hepatocytes (arrows) in a mousefed orally by CTB-GFP expressing plant. F) Section of the liver from amouse fed by wild-type (untransformed) plant. G) Section of the liverfrom a mouse fed by IFN-GFP expressing plant. H) GFP-immunoreactivity inthe spleen of mouse fed orally by CTB-GFP expressing plant. Arrowsindicate various cells with a higher GFP content. I) Section of thespleen from a mouse fed by wild-type (untransformed) plant. J) Sectionof the spleen from a mouse fed by wild-type (untransformed) plant. Scalebar for A-D_(—)50 μm, Scale bar for E-J_(—)25 μm.

FIG. 44. Immunohistochemistry of ileum, liver, and spleen tissues ofmice fed with CTBGFP expressing leaves or IFN-GFP expressing leaves orwild-type leaves. A) Shows a section of the intestine of a CTB-GFPtreated mouse. The arrows indicate CTB in the submucosa of theintestinal villi. B) Shows a section of mouse ileum fed with wild-typeplant, immunostained for CTB. C-F) Double staining for macrophage (red)and CTB (green) in mouse intestine and liver. C) Arrows show macrophagesin the submucosa of the intestine containing CTB, in a mouse fed withCTB-GFP expressing plant leaf material. The merged color is yellow. D)Arrows indicate F4/80-positive cells (macrophages, in red) in a mergedpicture in the intestine of a mouse fed with WT leaf material. E) Amerged picture showing double staining for macrophage (Kupffer cells)and CTB in mouse liver. Arrows show macrophages (red) in the liver. Nosign of CTB (green) was found in the liver of CTB-GFP fed mouse. F)Liver section of an IFN-GFP fed mouse used as a negative control forCTB. Macrophages are seen in red. G) F4/80 Ab was used as a marker ofmacrophages in the intestine. Arrows indicate macrophages, which haveentrapped GFP (yellow after merging the red and the green). Many of themacrophages are not associated with GFP. H) Many macrophages are seen inthe intestine of mouse fed with IFN-GFP expressing plant leaf material,which do not show GFP immunoreactivity. I, J) CD11c (red) and GFP(green) immunoreactivities in the mouse intestine. I) Arrows indicateCD11c (red, presumably dendritic cells, due to having a star shapemorphology) with internalized GFP (green), which can be seen in yellowcolor when the red and green channels were merged. J) Arrows indicateCD11c-positive cells in intestine of mice fed with IFN-GFP expressingplant leaf material. Scale bar for A and B_(—)25 μm. Scale bar forC-J_(—)50 μm.

FIG. 45: Transcriptional and translational analysis of the Cry2Aa2operon: A. Schematic representation of the orf1-orf2-cry2Aa2 operon intransgenic lines, including the aadA gene and the upstream Prrn promoter(P); upstream native chloroplast 16S ribosomal RNA gene with itsrespective promoter (Pan) and the trnI and trnA are shown. Arrowsrepresent expected transcripts and their respective sizes. B. RNAhybridized with the cry2A probe, loaded as follows: wt: wild typecontrol; lanes 1, 2 and 3: cry2Aa2 operon transgenic lines. Transcriptsof the cry2Aa2 operon are indicated by lowercase letters and correspondto the transcripts depicted in A. C. Relative heterologous transcriptabundance within each line hybridized with the cry2A probe. D.Transcript analysis showing RNA hybridization with the aadA probe,loaded as follows: wt: wild type control, lanes 1-3: cry2Aa2 operontransgenic lines. Transcripts of the cry2Aa2 operon are of sizes asdescribed for the cry2Aa2 probe; f is aadA/orf1/orf2 tricistron, 2,5knt. E. Heterologous transcript quantification for samples hybridizedwith the aadA probe. F. RNA hybridization using the orf1,2 probe.Samples were loaded in the same order as in D and predicted transcriptsizes correspond to those observed in D. G. Relative transcriptabundance within each transgenic line obtained by hybridization with theorf1,2 probeH. Western blot analysis using the Cry2Aa2 antibody. wt:wild type control; lanes 1 and 2: cry2Aa2 operon transgenic lines; lane3: positive control (Cry2Aa2 protein). The expected polypeptide of 65kDa is shown in both transgenic plants and the positive control. I.Western blot analysis using the ORF2 antibody. wt: wild type control;lanes 1 and 2: cry2Aa2 operon transgenic lines; lane 3: positive control(ORF2 protein). The expected polypeptide of 45 kDa is shown in bothtransgenic plants and the positive control.

FIG. 46: Polysome fractionation assays of the cry2Aa2 operon

A. RNA hybridized with the cry2A probe after fractionation through asucrose gradient. WT: wild type control, T: total RNA sample, lanes1-12: RNA collected from the different fractions of the gradient. Lowerfractions correspond to the bottom of the sucrose gradient (polysomalfractions). P: cry2Aa2 probe. c: transcript “c” (aadA-orf1-orf2-cry2Apolycistron) described in FIG. 1A. B. Same RNA blot after stripping andre-hybridizing with orf1,2 probe. Lane P is omitted because no orf1,2probe was loaded. C. Puromycin release and wild-type controls. Cry2Aa2samples were treated with puromycin before loading onto sucrosegradients, whereas an additional wild-type sample was loaded ontosucrose gradients and used as a negative control. RNA was hybridizedwith the aadA probe. The gel was loaded as follows: WT: wild-type RNA;T: total RNA; 1-11: RNA collected from the different fractions of thesucrose gradient and hybridized with the aadA probe. Lanes 12-16:wild-type RNA from fractions 2, 4, 6, 8 and 10 collected from thesucrose gradient. P: aadA probe. c: transcript “c” (aadA-orf1-orf2-cry2Apolycistron) described in FIG. 1A.

FIG. 47: Transcriptional and translational analysis of the hsa operons

A. Schematic representation of the hsa operons (rbs-hsa, 5′UTR-hsa,orf1-orf2-hsa) in transgenic lines, including the aadA gene and upstreamPrrn promoter (P); upstream native chloroplast 16S ribosomal RNA geneand promoter (Prrn) as well as trnI/trnA genes are shown. Arrowsrepresent expected transcripts and their respective sizes. B. RNAhybridization with the hsa probe. wt: wild type; lanes 1-3: rbs-hsatransgenic lines; lanes 4-6: 5′UTR-hsa transgenic lines; lanes 7-9:orf1,2-hsa transgenic lines. Lowercase letters correspond to thetranscripts predicted in A. C. Relative abundance of the transcriptsobtained with the hsa probe. D. mRNA transcripts hybridized with theaadA probe and loaded in the same order as in B. Transcripts a-icorresponded to the same transcripts observed in B, “k” corresponds tothe 16rrn/hsa polycistron (6,9 knt). E. Quantification of relativeheterologous transcript abundance obtained with the aadA probe. F. mRNAtranscripts of wild-type (wt) and orf1,2-hsa transgenic lines (lanes1-3) hybridized with the orf1,2 probe. G. Relative abundance obtainedfor the transcripts detected with the orf1,2 probe. H. Western blotanalysis using the HSA antibody. wt: wild type control. Lanes 1-2:RBS-hsa transgenic lines; lanes 3-4: 5′UTR-hsa transgenic lines. Lanes 5and 6 orf1,2-hsa transgenic lines. Lane 7: positive control (HSAprotein). Lane marked with (−) was left blank. All samples presented 66kDa and 132 kDa peptides, corresponding to the size of the HSA protein,and its dimeric form, respectively. I. Western blot analysis using theORF2 antibody. Lanes 1-2: orf1,2-hsa transgenic lines; lane 3: wild typecontrol; lane 4: positive control (ORF protein). 45 kDa ORF2 and 90 kDadimer are shown.

FIG. 48: ELISA analysis of the orf1-orf2-hsa transgenic line.

Total soluble protein content of young, mature and old leaf extracts ofthe orf1-orf2-hsa transgenic lines determined by ELISA analyses.Transgenic plants were subjected to the following light conditions: 4,8, and 16 hours of light, as well as total darkness.

FIG. 49: Transcriptional and translational analysis of the tps1 operon

A. Schematic representation of the tps1 operon in transgenic lines,including the aadA gene and upstream Prrn promoter (P). Upstream nativechloroplast 16S ribosomal RNA gene and promoter (Prrn) as well astrnI/trnA genes are shown. Arrows represent expected transcripts andtheir respective sizes. B. Northern blot analysis obtained byhybridization with the tps1 probe, loaded as follows: wt: wild typecontrol; lanes 1, 2 and 3: tps1 transgenic lines. Transcripts of thetps1 operon correspond to those depicted in A, indicated with lowercaseletters. C. Relative transcript abundance per transgenic line, obtainedwith the tps1 probe. D. RNA transcripts hybridized with the aadA probe,loaded as follows: wt: wild type control; lanes 1-3: tps1 transgeniclines. Transcript bands obtained for the tps1 operon are of sizes asdescribed for tps1 probe (B). D. Relative abundance of transcripts ineach sample after hybridization with the aadA probe. E. Western blotanalysis using the TPS1 antibody. Lane 1: positive control (TPS1protein); lane 2: wild type control; lane 3: tps1 transgenic line. Apolypeptide of 65 kDa was observed in the transgenic clone,corresponding to the expected size of the TPS1 protein, as observed inthe positive control.

FIG. 50: Transcriptional and translational analysis of the CTB operons

A. Schematic representation of the 5′UTR-ctb-gfp and RBS-ctb operon intransgenic lines, including the aadA gene and the upstream Prrn promoter(P); upstream native chloroplast 16S ribosomal RNA gene with itsrespective promoter (Prrn) and the trnI and trnA are also shown. Arrowsrepresent expected transcripts and their respective sizes. B. Northernblot analysis showing RNA hybridized with the CTB probe. Samples wereloaded as follows: wt: wild type control; lanes 1-3: 5′UTR-ctb-gfptransgenic lines; lanes 4-6: rbs-ctb transgenic lines. The transcriptsand respective sizes correspond to those indicated in A with lowercaseletters. C. Relative transcript abundance, within each line, of thetranscripts shown in B. D. RNA hybridization using the aadA probe, andloaded according to the following: M: molecular weight marker; wt: wildtype control; lanes 1-3: RBS-ctb transgenic lines. Lanes 4-6:5′UTR-ctb-gfp transgenic lines. Lanes marked with (—) were left blank.The transcripts observed correspond to the same as in B. E. Relativetranscript abundance, per line, for the transcripts shown in C. F.Western blot analysis of the RBS-CTB transgenic lines using anti-CTBantibody. Lanes 1-3: transgenic clones; lane 4: wild type control; lane5: positive control (CTB protein). CTB from transgenic lines is intrimeric form. E. Western blot analysis of the 5′UTR-ctb-gfp transgeniclines using the CTB antibody. Lane 1: Wild type control. Lanes 2-5:transgenic lines. Lane 6: positive control (CTB protein).

FIG. 51: Transcription of heterologous operons using the psbA 3′UTRprobe.

A. Northern blot analysis and corresponding quantification oftranscripts obtained from different HSA transgenic lines described inFIG. 3, as well as of the native psbA transcripts. The RNA gels wereloaded as follows: wt: wild-type. Lanes 1-3: RBS-HSA transgenic lines.Lanes 4-6: 5′UTR-HSA transgenic lines. Lanes 7-9: ORF-1,2-HSA transgeniclines. P: psbA 3′UTR probe. Lowercase letters correspond to the sametranscripts predicted in FIG. 3A. Transcript abundance was normalizedagainst the wild-type psbA, to which a value of 1 was assigned. B.Northern blot analysis and corresponding transcript quantification ofthe cry2Aa2 operon. Gel loading was as follows: wt: wild-type RNA. Lanes1-3: Cry2Aa2 transgenic lines. P: psbA 3′UTR probe. Lowercase letterscorrespond to transcripts predicted in FIG. 1A. Native psbA transcriptis indicated. Transcript abundance was normalized against the wild-typepsbA, to which a value of 1 was assigned. The low transcript abundanceof lane 1 is due to partial RNA degradation in the sample. C. RNA blotand transcript quantification of the transgenic TPS1 lines. The RNA gelwas loaded as follows: wt: wild-type. Lanes 1-3. TPS1 transgenic lines.P: psbA 3′UTR probe. Lowercase letters correspond to transcript sizesshown in FIG. 5A. Native psbA transcript is indicated Transcriptabundance was normalized against the wild-type psbA, showing a valueof 1. D. Northern blot analysis of the RBS-CTB and 5′UTR-CTB-GFPtransgenic lines. Samples were loaded as follows: wt: wild-type. Lanes1-3: RBS-CTB transgenic lines. Lanes 4-6: 5′UTR-CTB-GFP transgeniclines. P: psbA 3′UTR probe. Lowercase letters correspond to transcriptsshown in FIG. 6A. Transcripts a* and b* are similar in size to thenative psbA and therefore they cannot be distinguished from the nativetranscript. Because such transcripts were shown to be very abundant inFIG. 6B, and because of increase in transcript abundance in comparisonto the wild-type psbA transcript, it is assumed that such transcriptsare present. Transcript abundance was normalized against the wild-typepsbA, to which a value of 1 was assigned.

FIG. 52 Vector map and confirmation of transgene integration intochloroplast genome by PCR and Southern blotting. (a) Schematicrepresentation of pLD-VK1 vector with protective antigen gene (pagA),aadA (selectable marker), 5′UTR, and chloroplast flanking sequences forsite-specific integration with the primers 3P/3M and 5P/2M annealingsites within the native chloroplast genome and the schematic diagram ofexpected products from digestion of plants transformed with pLD-VK1. (b)Schematic diagram of expected products from digestion of wild-typeuntransformed plant. (c) Confirmation of site-specific transgenecassette integration by PCR using primers (3P/3M) to yield a 1.65-kbproduct. Lane 1, 1-kb DNA ladder; lane 2, wild type; lanes 3 to 6,pLD-VK1 transgenic lines; lane 7, positive control (interferontransgenes).

FIG. 53. Immunoblotting analysis and quantification of PA expressed inchloroplast of transgenic plants (pLD-VK1) in T0 generation. (a)Immunoblotting demonstrating the expression of PA in transgenic plantcrude extracts. Lane 1, wild type; lane 2, 100-ng standard; lane 4,transgenic line 5; lane 6, transgenic line 7; lane 8, transgenic line 8;lanes 3, 5, and 7, empty. (b) Expression levels in percent TSP ofPA-expressing leaves (young, mature, and old) under normal andcontinuous illumination observed for 0 to 7 days.

FIG. 54. Purification of PA by affinity chromatography from the crudeextracts of plant leaves expressing PA. (a) Coomassie staining of theproteins in crude extract and purified protein: Lane 1, protein plusprecision ladder; lane 2, wild-type leaf crude extract; lane 3, crudeextract of transgenic plant expressing PA; lanes 4 and 5, purifiedchloroplast-derived PA; lane 6, flowthrough collected duringpurification. (b) Lane 1, ladder; lane 3, concentrated protein; lane 5,purified protein (before concentrating); lanes 2 and 4, overflow fromlane 3.

FIG. 55. Functional analysis of PA with macrophage cytotoxicity assay.The cytotoxicities of various PA preparations for mouse macrophageRAW264.7 cells were assayed in the presence of LF. Samples that werediluted serially were as follows: crude extract of plant leavesexpressing PA with His tag, wild-type (WT) plant leaf crude extract,20-μg/ml stock of purified chloroplast-derived PA, 20-μg/ml stock ofpurified PA derived from B. anthracis, and plant protein extractionbuffer.

FIG. 56. IgG antibody titers and toxin neutralization assay titers inserum samples obtained from mice after third and fourth doses. (a)Comparison of immune responses in serum samples of mice administeredsubcutaneously with chloroplast-derived PA (CpPA) with adjuvant (column1), chloroplast-derived PA (CpPA) alone (column 2), Std-PA derived fromB. anthracis with adjuvant (column 3), Std-PA alone (column 4), PA plantleaf crude extract with adjuvant (column 5), wild-type plant leaf crudeextract with adjuvant (column 6), and unimmunized mice (column 7). (b)Toxin neutralization titers of sera collected from the mice on day 43 ofpost-initial immunization. Each symbol represents average EC50 fromthree replicate assays of a single mouse serum. CHL, chloroplast; ADJ,adjuvant; B.A., B. anthracis; WT, wild type. (c) Toxin neutralizationassays of serum samples collected from the mice on day 155 ofpost-initial immunization. Each symbol represents the average EC50 fromthree replicate assays of a single mouse serum.

FIG. 57. Toxin challenge of the mice with systemic anthrax lethal toxin.Shown is survival over time for different groups of mice after challengewith a 150-μg dose of lethal toxin. IP, intraperitoneal; CHLPST,chloroplast; ADJ, adjuvant; WT, wild type.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of molecular biology. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed herein. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York (1982) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork (1989); Methods in Plant Molecular Biology, Maliga et al, Eds.,Cold Spring Harbor Laboratory Press, New York (1995); Arabidopsis,Meyerowitz et al, Eds., Cold Spring Harbor Laboratory Press, New York(1994) and the various references cited therein.

Methods, vectors, and compositions for transforming plants and plantcells are taught for example in WO 01/72959; WO 03/057834; and WO04/005467. WO 01/64023 discusses use of marker free gene constructs.

Proteins expressed in accord with certain embodiments taught herein maybe used in vivo by administration to a subject, human or animal in avariety of ways. The pharmaceutical compositions may be administeredorally or parenterally, i.e., subcutaneously, intramuscularly orintravenously. Thus, this invention provides compositions for parenteraladministration which comprise a solution of the fusion protein (orderivative thereof) or a cocktail thereof dissolved in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., water, buffered water, 0.4% saline, 0.3% glycerineand the like. These solutions are sterile and generally free ofparticulate matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. The concentration of fusion protein (or portion thereof)in these formulations can vary widely depending on the specific aminoacid sequence of the subject proteins and the desired biologicalactivity, e.g., from less than about 0.5%, usually at or at least about1% to as much as 15 or 20% by weight and will be selected primarilybased on fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected.

Oral vaccines produced by embodiments of the present invention can beadministrated by the consumption of the foodstuff that has beenmanufactured with the transgenic plant producing the antigenic likeparticles. The edible part of the plant is used as a dietary componentwhile the vaccine is administrated in the process.

To evaluate the antigenicity of the expressed antigens, the level ofimmunoglobulin A in feces or immunoglobulin G in serum is measured,respectively, after test animals has been immunized with the antigenembodiments of the present invention by oral administration orperitoneal injection. The ability to elicit the antibody formation ismeasured by Enzyme-linked immunosorbent assay. In addition, the directconsumption of the transgenic plant producing the antigen induces theformation of antibodies against the specific antigen.

The vaccines of certain embodiments of the present invention may beformulated with a pharmaceutical vehicle or diluent for oral,intravenous, subcutaneous, intranasal, intrabronchial or rectaladministration. The pharmaceutical composition can be formulated in aclassical manner using solid or liquid vehicles, diluents and additivesappropriate to the desired mode of administration. Orally, thecomposition can be administered in the form of tablets, capsules,granules, powders and the like with at least one vehicle, e.g., starch,calcium carbonate, sucrose, lactose, gelatin, etc. The preparation mayalso be emulsified. The active immunogenic ingredient is often mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredient. Suitable excipients are, e.g., water,saline, dextrose, glycerol, ethanol or the like and combination thereof.In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, or adjuvants which enhance the effectiveness of the vaccines.The preparation for parental administration includes sterilized water,suspension, emulsion, and suppositories. For the emulsifying agents,propylene glycol, polyethylene glycol, olive oil, ethyloleate, etc. maybe used. For suppositories, traditional binders and carriers may includepolyalkene glycol, triglyceride, witepsol, macrogol, tween 61, cocoabutter, glycerogelatin, etc. In addition, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate and the like can be used as excipients.

Antigen(s) may be administered by the consumption of the foodstuff thathas been manufactured with the transgenic plant and the edible part ofthe plant is used directly as a dietary component while the vaccine isadministrated in the process.

The vaccine may be provide with the juice of the transgenic plants forthe convenience of administration. For said purpose, the plants to betransformed are preferably selected from the edible plants consisting oftomato, carrot and apple, which are consumed usually in the form ofjuice.

The vaccination will normally be taken at from two to twelve weekintervals, more usually from three to hive week intervals. Periodicboosters at intervals of 1-5 years, usually three years, will bedesirable to maintain protective levels of the antibodies. It will bedesirable to have administrations of the vaccine in a dosage range ofthe active ingredients of about 100-500 μg/kg, preferably 200-400 μg/kg.

According to one embodiment, the subject invention relates to a vaccinederived from a plant transformed to express antigenic proteins capableof producing an immune response in a subject (human or non-humananimal).

According to another embodiment, the subject invention pertains to atransformed chloroplast genome that has been transformed with a vectorcomprising a heterologous gene that expresses a peptide as disclosedherein.

Of particular present interest is a transformed chloroplast genome thathas been transformed with a vector comprising a heterologous gene thatexpresses a peptide antigenic for rotavirus, hepatitis C or Anthrax. Ina related embodiment, the subject invention pertains to a plantcomprising at least one cell transformed to express a peptide asdisclosed herein.

Accordingly, in one embodiment, a vaccine pertains to an administratablevaccine composition that comprises an antigen having been expressed by aplant and a plant remnant. A plant remnant may include one or moremolecules (such as, but not limited to, proteins and fragments thereof,minerals, nucleotides and fragments thereof, plant structuralcomponents, etc.) derived from the plant in which the antigen wasexpressed. Accordingly, a vaccine pertaining to whole plant material(e.g., whole or portions of plant leafs, stems, fruit, etc.) or crudeplant extract would certainly contain a high concentration of plantremnants, as well as a composition comprising purified antigen that hasone or more detectable plant remnants.

Reference to specific polypeptide sequences herein (such as but notlimited to, CTB, proinsulin, interferon alpha, GFP, NSP4, HCV NS3protein, yeast trehalose phosphate synthase, human serum albumin,Cry2Aa2 protein, and/or protective antigen relate to the full lengthamino acid sequences as well as at least 12, 15, 25, 50, 75, 100, 125,150, 175, 200, 225, 250 or 265 contiguous amino acids selected from suchamino acid sequences, or biologically active variants thereof.

Variants which are biologically active, refer to those, in the case ofimmunization, confer an ability to induce serum antibodies which protectagainst infection against the pathogen from which polypeptide isderived, or, in the case of desiring the native function of the protein,is a variant which maintains the native function of the protein.Preferably, naturally or non-naturally occurring polypeptide variantshave amino acid sequences which are at least about 55, 60, 65, or 70,preferably about 75, 80, 85, 90, 96, 96, or 98% identical to thefull-length amino acid sequence or a fragment thereof. Percent identitybetween a putative polypeptide variant and a full length amino acidsequence is determined using the Blast2 alignment program (Blosum62,Expect 10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acidsubstitutions, insertions, or deletions Amino acid substitutions aredefined as one for one amino acid replacements. They are conservative innature when the substituted amino acid has similar structural and/orchemical properties. Examples of conservative replacements aresubstitution of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of polypeptide can be found using computerprograms well known in the art, such as DNASTAR software. Whether anamino acid change results in a biologically active LecA polypeptide canreadily be determined by assaying for native activity, as described forexample, in the specific Examples, below.

Reference to genetic sequences herein refers to single- ordouble-stranded nucleic acid sequences and comprises a coding sequenceor the complement of a coding sequence for polypeptide of interest.Degenerate nucleic acid sequences encoding polypeptides, as well ashomologous nucleotide sequences which are at least about 50, 55, 60, 65,60, preferably about 75, 90, 96, or 98% identical to the cDNA may beused in accordance with the teachings herein polynucleotides. Percentsequence identity between the sequences of two polynucleotides isdetermined using computer programs such as ALIGN which employ the FASTAalgorithm, using an affine gap search with a gap open penalty of −12 anda gap extension penalty of −2. Complementary DNA (cDNA) molecules,species homologs, and variants of nucleic acid sequences which encodebiologically active polypeptides also are useful polynucleotides.

Variants and homologs of the nucleic acid sequences described above alsoare useful nucleic acid sequences. Typically, homologous polynucleotidesequences can be identified by hybridization of candidatepolynucleotides to known polynucleotides under stringent conditions, asis known in the art. For example, using the following wash conditions:2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, roomtemperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once,30 minutes; then 2×SSC, room temperature twice, 10 minutes eachhomologous sequences can be identified which contain at most about25-30% basepair mismatches. More preferably, homologous nucleic acidstrands contain 15-25% basepair mismatches, even more preferably 5-15%basepair mismatches.

Species homologs of polynucleotides referred to herein also can beidentified by making suitable probes or primers and screening cDNAexpression libraries. It is well known that the Tm of a double-strandedDNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner etal., J. Mol. Biol. 81, 123 (1973). Nucleotide sequences which hybridizeto polynucleotides of interest, or their complements following stringenthybridization and/or wash conditions also are also usefulpolynucleotides. Stringent wash conditions are well known and understoodin the art and are disclosed, for example, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2^(nd) ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination oftemperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a polynucleotide of interest or thecomplement thereof and a polynucleotide sequence which is at least about50, preferably about 75, 90, 96, or 98% identical to one of thosenucleotide sequences can be calculated, for example, using the equationof Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(% formamide)−600/l),

where l=the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4×SSC at 65° C., or 50%formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

Relevant articles on genetic sequences is provided: proinsulin(Brousseau et al., Gene, 1982 March; 17(3):279-89; Narrang et al, Can JBiochem Cell Biol. 1984 April; 62(4):209-16; and Georges et al, Gene 27(2), 201-211 (1984)); GFP (Prasher et al., Gene 111, 229-233, (1992));protective antigen (Welkos et al., Gene. 1988 Sep. 30; 69(2):287-300);alpha interferon (Strausberg et al., Proc. Natl. Acad. Sci. U.S.A. 99(26), 16899-16903 (2002)); rotavirus (Kirkwood et al., Virus Genes,Volume 19, Issue 2, October 1999, Pages 113-122); hepatitis C NS3(Lodrini et al., J Biol Regul Homeost Agents. 2003 April-June;17(2):198-204); and CTB (Shi et al, Sheng Wu Hua Hsuch Tsa Chih 9 (No.4), 395-399 (1993).

Example 1 Expression of Cholera Toxin B Subunit-Proinsulin FusionProtein in Transgenic Tobacco Chloroplasts as a Treatment AgainstDevelopment of Type-1 Autoimmune Diabetes

Oral administration of disease specific autoantigens have beendemonstrated to delay or even prevent the onset of disease symptoms,referred to as tolerance. The inventor has produced a Nicotiana tabacumcv. petit Havana chloroplast transgenic lines that expresses a choleratoxin B subunit (from Vibrio Cholerae)-human proinsulin (a and b chain)fusion protein, designated CTB-Pris. This approach has been previouslydemonstrated by nuclear expression in potato tubers, to prevent theonset of insulin-dependent diabetes mellitus (IDDM) in NOD mice, whendelivered orally. The pLD-PW contains the CTB-Pris gene cloned into theuniversal chloroplast transformation vector pLD-ctv in which the 16SrRNA promoter drives the aadA gene selectable marker, which confersresistance to spectinomycin; the psbA 5′ untanslated region (UTR)enhanced translation of CTB-Pris in the presence of light and the psbA3′UTR conferred transcript stability. The trnI and trnA homologousflanking sequences facilitated site-specific integration of transgenesinto the tobacco chloroplast genome. Site-specific integration wasdemonstrated by PCR and Southern blot analysis with probes for both CTBand Pris. Western Blot analysis has demonstrated the presence ofabundant CTB-Pris in transgenic plants with both CTB polyclonal andproinsulin monoclonal antibodies. Southern blot analysis has alsoconfirmed that homoplasmy had been achieved in the T0 generation. Thesechloroplast transgenic lines grew slowly although their appearance wasnormal. Quantification studies are conducted as well as animal studieson NOD mice in order to determine the ED50 for prevention of the onsetof insulin-dependent diabetes mellitus. Techniques for planttransformation and processing and uses of expressed protein arediscussed in WO 01/72959.

Diabetes is a disease in which the body does not produce or properlyutilizeinsulin. Type 1 diabetes results from the autoimmune destructionof insulin-producing cells and a corresponding failure to produceadequate insulin. In 2002, the American Diabetes Association estimatedthat 18.2 million people in the United States, or 6.3% of the totalpopulation, have diabetes, with more than $120 billion in treatmentcosts each year. Complications that can arise from diabetes include:heart disease, nephropathy, retinopathy, neuropathy, hypertension, footcomplications, skin problems, gastroparesis, and depression. In 2002,diabetes was the sixth leading cause of death in the U.S. contributingto 213,062 deaths. The only currently accepted form of treatment is theadministration of recombinant insulin, which serves to temporarilyreplace the missing insulin in diabetic patients. Therefore, it isessential to find a prevention and cure for this dreadful disease.

Insulin is secreted by pancreatic β-cells and is regulated by aglucose-sensing system. The insulin polypeptide is made up of an A chain(21 amino acids) and a B chain (30 amino acids) that are bound togethervia disulfide bonds across cysteine residues. It is initially translatedin the endoplasmic reticulum as preproinsulin, processed into proinsulinthat is trafficked to the secretory granules of the β-cell, and finallyprocessed into C-peptide and mature insulin (reviewed in Halban 1991).The function of C peptide, which is part of proinsulin prior toprocessing, has remained a mystery until recently, when human trialshave demonstrated that the proinsulin C-peptide stimulates theactivities of Na+,K+-ATPase and endothelial nitric oxide synthase, bothof which are enzyme systems of importance for nerve function and knownto be deficient in Type 1 diabetes (Ekberg et al., 2003). The majordestruction of β-cells occurs predominantly from autoreactiveT-cytotoxic cells (Nagata et al., 1994) and T-helper 1 cells (Ploix etal., 1999) reactive to β-cell autoantigens such as insulin.

Biopharmaceutical proteins expressed in plant cells should reduce theircost of production, purification, processing, cold storage,transportation and delivery. Integration of transgenes via the nucleargenome may have a few disadvantages. The chloroplast genetic engineeringapproach overcomes such concerns of transgene containment (Daniell2002), low levels of transgene expression, gene silencing, positioneffect, pleiotropic effects, and presence of antibiotic resistant genesor vector sequences in transformed genomes (Daniell et al. 2002,2004a-c, 2005a,b; Grevich & Daniell, 2005). This approach has beensuccessfully used in our laboratory to confer desired plant traits(Daniell et al. 1998; Kota et al. 1999; DeGray et al. 2001; Lee et al.2003; Kumar et al. 2004a; Ruiz et al. 2003). Employing knowledge gainedthrough these studies, we have demonstrated expression and assembly ofseveral vaccine antigens, including the cholera toxin B subunit (CTB,Daniell et al. 2001), the F1˜V fusion antigen for plague (Singleton2003; Daniell et al. 2005b), the 2L21 peptide from the canine parvovirus(CPV, Molina et al. 2004), the anthrax protective antigen (PA, Watson etal. 2004), and the NS3 protein as a vaccine antigen for hepatitis C(Bhati 2004). Cytotoxicity measurements in macrophage lysis assaysshowed that chloroplast-derived anthrax protective antigen (PA) wasequal in potency to PA produced in B. anthracis. It was reported thatone acre of land should produce 360 million doses of a purified vaccinefree of the bacterial toxins (Koya et al. 2005) because of the largeyield of biomass. In addition to its use for the hyper-expression ofvaccine antigens, transgenic chloroplasts have been used in our lab forthe production of valuable therapeutic proteins, such as humanelastin-derived polymers for various biomedical applications (Guda etal. 2000); human serum albumin (Fernandez-San Millan et al. 2003);magainin, a broad spectrum topical agent, systemic antibiotic, woundhealing stimulant and a potential anticancer agent (DeGray et al. 2001);various interferon α proteins (Daniell et al. 2004b, 2005b); andinsulin-like growth factor 1 (Ruiz 2002). Several other laboratorieshave expressed other therapeutic proteins, including human somatotropin(Staub et al. 2000) and interferon γ-GUS fusion proteins (Leelavathi &Reddy 2003), and the C-terminus of Clostridium tetani (Tregoning et al.2003) in transgenic chloroplasts. The successful expression and assemblyof complex multi-subunit proteins has demonstrated that chloroplastscontain the machinery that allows for correct folding and disulfide bondformation, resulting in fully functional proteins (Daniell et al. 2004b,2005b).

Oral delivery of biopharmaceutical proteins expressed in plant cellsshould reduce their cost of production, purification, processing, coldstorage, transportation and delivery. However, poor intestinalabsorption of intact proteins is a major challenge. To overcome thislimitation, we investigated the concept of receptor-mediated oraldelivery of transgenic proteins. Therefore, the transmucosal carriercholera toxin B-subunit and green fluorescent protein (CTB-GFP),separated by a furin cleavage site, was expressed via the tobaccochloroplast genome. Following oral administration of CTBGFP expressingleaf material to mice, GFP was observed in the mice intestinal mucosa,liver and spleen in fluorescence and immunohistochemical studies, whileCTB remained in the intestinal cell (Limaye et al., 2006). This reportof receptor-mediated oral delivery of a foreign protein into thecirculatory system brings the delivery of human therapeutic proteins onestep closer to realization. Transformation of non-green tissue plastids(Kumar et al. 2004 a,b; Daniell et al., 2005a) was recently achieved,further facilitating the oral delivery of therapeutic proteins.

The non-obese diabetic (NOD) mouse is a useful animal model for researchin human diabetes. About 60%-75% of NOD mice become diabetic by 40 weeksof age (Homann et al., 1999a). These mice show signs of insulitis due tolymphocytic infiltration of the endocrine part of the pancreas, whichleads to decreased production of insulin and increased blood sugar withits consequent pathologies. In this study, we examine the effect of oraladministration of chloroplast-derived proinsulin conjugated to CTB forthe induction of oral tolerance towards insulin. Oral administration ofsmall quantities of CTB-Proinsulin to NOD mice leads to its uptake byintestinal epithelial cells via the GM1 receptor. These cells then passthe antigen (proinsulin) to the underlying antigen presenting cells(APCs), such as macrophages or dendritic cells. These cells in turnactivate lymphocytes to up-regulate the Th2 response, leading to theproduction of immune-suppressing cytokines such as interleukins 4 (IL-4)and 10 (IL-10), which suppress (reduce) the immune attack against theendocrine insulin-producing β-cells of the Langerhans islets of thepancreas.

Material and Methods Vector Construction

The human proinsulin gene was synthesized by a method that utilized fouroverlapping oligos with a low annealing temperature (50° C.). Theproducts were then used as templates for high annealing temp (65° C.)primers, thus synthesizing the required 258 bp gene (Protocol fromProdromou and Pearl, 1992). The PCR product was then subsequently clonedinto the PCR 2.1 vector and the sequence verified. The psbA promoter and5′ untranslated region (UTR) was amplified from the tobacco chloroplastgenome, followed by sub-cloning, and sequence verification. Thepromoter-5′UTR fragment was then spliced together with the cholera toxinB-subunit (CTB) and human proinsulin by a process that utilizes fourprimers (Splicing by Overlap Extension, Horton et al., 1989). Thus, theconstruct now contained the 5′UTR-CTB, and a GPGP(glycine-proline-glycine-proline) hinge region introduced by mutagenesisto allow for the proper folding of each protein by reducing sterichindrance, followed by human proinsulin; the final construct was termed5CP. Following SalI/NotI digestion to release the fusion gene ofinterest, it was ligated into the pLD-ctv chloroplast transformationvector. The 5CP insert was ligated into the chloroplast transformationvector, pLD-ctv which was developed previously by the Daniell laboratory(Daniell et al., 1998; 2004c).

Bombardment and Selection of Transgenic Plants

The Bio-Rad PDS-1000/He biolistic device was used to bombardpLD-CTB-Pins onto sterile Nicotiana tabacum cv. Petit Havana tobaccoleaves, on the abaxial side as has been previously described (Daniell,1997; Daniell et al., 2004c). The bombarded leaves were incubated in thedark for 24 hours and then placed on shoot inducing media (RMOP)containing 500 μg/ml spectinomycin for two rounds of selection. This wasfollowed by another round of selection on MSO, a root-inducing mediumwhich contained 500 μg/ml spectinomycin.

Southern Blot Analysis

Total plant DNA was digested with AflIII, separated on a 0.7% agarosegel at 45V for 8 hours, and then transferred to a nylon membrane. ThepUC-CT vector DNA was digested with BamHI and BglII to generate a 0.8 kbprobe which was used as a flanking probe (Daniell et al., 2004c) andpLD-CTB-Pins was digested with MfeI and NotI to generate the 0.36 kbgene specific probe. After labeling the probe with P32, hybridization ofthe membranes was done using QUICK-HYB hybridization solution andprotocol (Stratagene, La Jolla, Calif.).

Western Blot Analysis

Approximately 100 mg of leaf tissue was ground in liquid nitrogen andresuspended in 500 μl of plant extraction buffer (0.1% SDS, 100 mM NaCl,200 mM Tris-HCl pH 8.0, 0.05% Tween 20, 400 mM sucrose, 2 mM PMSF).After centrifugation at 13,000 rpm for 5 minutes, the supernatantcontaining the soluble extracted protein was collected. The plantextract along with the sample loading buffer were boiled, and then runon a 13% SDS-PAGE gel for 40 mins at 50V and then 2 hours at 80V. Theprotein was then transferred to nitrocellulose membrane for 1 hour at85V. After blocking the membranes with PTM (1×PBS, 0.05% Tween 20, and3% dry milk) for 1 hour, mouse anti-proinsulin monoclonal antibody(Amersham Pharmacia) at a 1:20,000 dilution was added and incubated for2 hours. Goat anti-mouse IgG antibody conjugated to horseradishperoxidase (Sigma) at a 1:15,000 dilution was used as a secondaryantibody and incubated for 1.5 hours.

Quantification via ELISA

Approximately 100 mg of leaf tissue was ground in liquid nitrogen andresuspended in 500 μl of plant extraction buffer (15 mM Na2CO3, 35 mMNaHCO3, 3 mM NaN3, pH 9.6, 0.1% Tween 20 and 5 mM PMSF). Using the TotalProinsulin ELISA Kit (Linco Research, St Charles, Mo.) and followingmanufactures instructions, the Insulin present in the leaf wasquantified. Ninety-six well plates were read on a plate reader (DynexTechnologies) at 450 nm.

Animal Studies

Four week old female non-obese diabetic (NOD) mice were purchased fromThe Jackson Laboratory (Bar Harbor, Me.). Mice were kept in the UCF WildAnimal Facility under normal light/dark cycle conditions and had accessto food and water ad lib. Treatment by means of oral administration ofCholera toxin B subunit-Proinsulin (CTBPins) expressing transgenic orcontrol plant leaf material began when animals were 5 weeks old, toallow the mice one week to acclimate to the facility. Mice were dividedinto the following groups: group 1 was fed untransformed plant leafmaterial; group 2 was fed transgenic plant leaf expressing Cholera toxinB subunit conjugated to GFP (CTB-GFP); group 3 was fed transgenic plantleaf expressing interferon conjugated to GFP (IFNGFP); and group 4 wasfed CTB-Pins expressing transgenic plant leaf. Each group contained fiveanimals, except the CTB-Pins group, which contained seven. Mice were fed8 mg of the specified ground plant leaf material once a week for 7weeks. The animals were sacrificed at 12 weeks of age, the pancreas andother tissues were collected, and both blood and urine glucose levelswere measured.

Blood and Urine Glucose Levels

Blood and urine glucose levels were measured for two consequent weeks(11 and 12 weeks old) with urinary glucose test strips (Clinistix andDiastix, Bayer), and blood glucose was measured by bleeding from eitherthe tail vein or the retro-bulbar vein (at week 12, before sacrificing)by blood glucose analyzer (Boehringer Mannheim). Blood glucose levelsover 250 mg/dl were considered diabetic (Arakawa et al, 1998).

Histochemistry for Lymphocytic Infiltration and Insulitis

Following the 7 week treatment, mice were sacrificed and perfusedtranscardially with 10 ml of PBS followed by 50 ml of 4%paraformaldehyde in PBS. Fresh frozen sections of the pancreas werecollected (Samsam et al., 2003). The pancreas was removed, post-fixedovernight, and then cryo-protected by serially passing through 10%, 20%and 30% sucrose solutions in PBS. The pancreatic tissue was thenimmersed in Tissue Tek freezing medium (Vector labs) and frozen inliquid nitrogen-cooled isomethylbuthane (isopathane, Sigma). Tenmicrometer (nm) thick frozen sections of the pancreas were then preparedusing a cryostat. Pancreas cryosections were stained with Hematoxillinand Eosin, dehydrated in serial graded alcohol solutions, and the slideswere cover slid.

Insulitis levels were measured based on the extent of the lymphocyteinfiltration of the islets of Langerhans. At least 50 sections peranimal were scored, the degree of insulitis was scored based on a 5level scale ranging from 1-5, where score 1 is a normal islet with nosign of T-cell infiltration, and score 5 indicates increasing ofinsulitis.

Immunohistochemistry for Insulin, Caspase-3, Interleukin (IL) 4 and IL10

Immunohistochemistry for the localization of insulin, caspase-3 (a finalmolecule of apoptosis), and the immunosuppressive cytokines IL4 and IL10were performed on the pancreas cryosections. Sections were blocked with10% BSA (bovine serum albumin) containing 0.3% Triton-X 100.

Polyclonal guinea pig anti-insulin, polyclonal rabbit anti-caspase-3,rat monoclonal anti-IL4 and anti-IL10 primary antibodies (Invitrogen)were diluted at a concentration of 1:300 in 1% BSA in PBS containing0.3% Triton-X. Fluorescent conjugated secondary antibodies were goatanti-guinea pig-Alexa Fluor 488 (green), goat anti-rabbit-Alexa Fluor555 (red), and goat anti-rat Alexa Fluor 555 (red, Invitrogen).

Antibody Titer

Serum and intestinal antibodies were assayed for the presence ofanti-insulin and anti-CTB antibodies using colorimetric ELISA methods.Ninety-six well plates were coated with either CTB or human insulin(Sigma). Serial dilutions of serum or supernatants of fecal pelletscollected from the different animal groups were added to the coatedmicrotiter plate wells. Secondary antibodies were horseradish peroxidase(HRP)-conjugated anti-mouse IgG2a, IgG1, or IgA antibodies (BDPharmingen, USA) at a concentration of 1:3000 in PBS containing 0.1%Tween-20 and 3% milk powder. The plates were washed with 200 μl of PBS,and the substrate tetra-methyl benzidine (TMB) was added to the wellsand incubated in the dark at 37° C. for 20 minutes. The reaction wasstopped by adding 50 μl of H2SO4 and the plates were read on a platereader (Dynex Technologies) at 450 nm.

Results

Vector Construction of pLD-5′UTR-CTB-Human Proinsulin (5CP)

The CTB-Pris fusion gene was inserted into the chloroplasttransformation vector pLD-ctv for homologous recombination into thetobacco chloroplast genome. The pLD vector contains the trnI and trnAflanking sequences utilized to facilitate homologous recombination intothe inverted repeat region of the tobacco chloroplast genome. The 5CPconstruct was expressed under the control of the psbA 5′UTR/promoter inorder to achieve hyper-expression as previously demonstrated (FernandezSan-Millan et al., 2003; Daniell et al., 2004c; Dhingra et al., 2004).The aadA gene confers resistance to spectinomycin in order to select fortransformed shoots (Goldschmidt-Clermont 1991) and is regulated by the16S rRNA promoter. The 3′UTR located at the 3′ end of the introducedgene confers transcript stability (Stern and Gruissem 1987). The pLDvector also possesses the chloroplast origin of replication(autonomously replicating sequence) located within the trnI region(Kunnimalaiyaan and Nielsen, 1997) which promotes replication of theplasmid following bombardment (FIG. 1). Crude extracts from E. coliclones that contained the 5′UTR-CTB-proinsulin insert was subjected toSDS-PAGE/immunoblotting, along with untransformed E. coli that served asa negative control. Following immunoblot detection with the insulinantibody, the correct size (˜22 kDa) CTBPro insulin fusion protein wasconfirmed (FIG. 2, Lane 1).

Analysis of the Transgenic Chloroplast Genome Reveals Homoplasmy

The chloroplast transgenic lines were subjected to Southern blotanalysis in order to confirm site-specific integration and to determinewhether they were homoplasmic or heteroplasmic. Homoplasmy is achievedwhen all the copies of the genome within the chloroplast have stablyintegrated transgenes. The gene-specific probe (CTB-proinsulin) that wastaken from the pLD-5CP vector by MfeI/NotI digestion (FIG. 3A) (360 bp)bound to the proper transgenic plant fragment but not wild-type plantfragment (FIG. 3C) following digestion of transgenic plant DNA withAflIII (FIG. 3B). This indicates that the gene of interest wasintegrated into the correct region within the chloroplast genome and theuntransformed plant DNA showed no such hybridization. The flankingsequence probe which contains the region of the trnI and trnA genes wasobtained by digestion of pUC-ct vector by BglII/BamHI digestion (FIG.3A). Chloroplast transgenic and untransformed plant DNA was digestedwith AflIII (FIG. 3B). Upon hybridization with the flanking sequenceprobe, transformed chloroplasts should yield a 6.4 kb fragment;untransformed plants, a 4.2 kb fragment. If the 4.2 kb fragment is notseen within the transgenic line, all the chloroplast genomes carry thegene of interest, and homoplasmy has been attained within our currentlimit of detection. Most of the lines tested showed only the 6.4 kbfragment when hybridized to transgenic plant DNA (FIG. 3D), indicatingthat homoplasmy was indeed achieved within limits of detection.

CTB-Proinsulin Pentamers were Assembled in Transgenic Chloroplasts

Immunoblots showed the presence of ˜22 kDa fusion protein in thechloroplast transgenic lines. The formation of monomers, dimers,trimers, tetramers, and pentamers of the CTB-Pins fusion protein wasalso observed (FIG. 2). A similar banding pattern was observed byimmunodetection with both the proinsulin monoclonal antibody (FIG. 2)and the CTB polyclonal antibody (data not shown). Quantification of thefusion protein on western blots was performed by comparing plant sampleswith a known quantity of purified CTB and reading them on an alphaimager by spot densitometry. Three different transgenic lines were foundto contain 358 μg, 270 μg and 364.8 μg of CTB-proinsulin per 100 mg ofleaf tissue, or approximately 30% of total soluble protein (tsp).Quantification by Total Proinsulin ELISA Kit of frozen plant tissuerevealed that the same transgenic lines contained 145 μg, 95 μg and 194μg of CTB-proinsulin per 100 mg of frozen leaf tissue, for a maximum of13.8% tsp. Such variation could be due to the use of fresh versus frozenplant material for these assays or differences in sample preparation orgrowth conditions.

Blood Glucose Levels of NOD Mice Treated with CTB-Human Proinsulin wereLowered

We divided the NOD mice into four groups fed once a week for seven weeksbeginning at week 5. Each group differed only by the plant material theywere fed. We fed one group untransformed plant material to control forthe potential effects of plant material alone. One group receivedtransgenic plant material expressing Cholera toxin B subunit-GFP fusionprotein (CTB-GFP; Limaye et al, 2006) to assess the effects of highlevels of conjugated CTB. Another group received transgenic plantmaterial expressing interferon alpha 5 conjugated to GFP (IFN-GFP) asyet another control of GFP without CTB. The last group receivedCTB-Pins, which we hypothesized would protect against the onset ofinsulitis in these mice.

Blood and urine glucose levels of the treated NOD mice were measuredtwice, at weeks 6 and 7. The blood glucose values of all groups in thisstudy at both time points tested were below 200 mg/dl, and thereforeconsidered to be within a normal, nondiabetic range. This was not anunforeseen possibility, since NOD mice typically do not develop highblood glucose until 12-15 weeks age (Arakawa et al, 1998). However, theCTB-Pins treated animals tended to have lower blood glucose values thanthe control groups (data not shown). Likewise, urine glucose values werealso within normal limits.

Lymphocytic Infiltration of the Endocrine Part of Pancreas (Insulitis)

Insulitis is characterized by lymphocytic infiltration of the pancreaticislets, accompanied by the secretion of proinflammatory cytokines, whichleads to the destruction of the pancreatic islets, including the insulinproducing beta cells. We collected pancreata from twelve week old NODmice from the different treatment groups to assess the degree ofinsulitis. Representative sections prepared from each treatment groupshowed that oral administration of transgenic plant material expressingCTB-Pins led to much less destructive cellular infiltration of thepancreatic islets as compared to the other experimental groups (FIG. 4).

To quantify and compare the insulitis of each treatment group, cellularinfiltrations were scored blindly according to the following: no isletcellular infiltration or pre-islet infiltration were scored 1, minimalinfiltrations were scored 2, moderate infiltrations were scored 3, andsevere infiltrations were scored 4 (FIG. 5). When more than 80% of theislets were infiltrated, the score was 5 (FIG. 5). Accordingly, fiftysections per animal were analyzed and the average score indicated thatthe pancreata from NOD mice administered CTB-Pins had minimal cellularinfiltration, and this reduction in cellular infiltration issignificantly less than all other treatment groups (FIG. 6).

Preservation of the Insulin Producing β-Cells Following Oral Delivery ofCTB-Pins

We next wanted to determine if the remaining β-cells represented in thepancreata of the different treatment groups were apoptotic. Becausecellular infiltration can lead to apoptosis, this could be used as ahallmark to study type 1 diabetes. Therefore, we labeled sections withinsulin and caspase-3, a known marker for apoptosis (Riedl & Shi, 2004).We found that the β-cells from NOD mice administered CTB-Pins rarelyexpressed caspase-3, suggesting that apoptosis was prevented in thesecells (FIG. 7). In the other experimental groups, even the very fewremaining insulin-producing β-cells expressed activated caspase-3,suggesting that they were undergoing apoptosis (FIG. 7).

Induction of Th2 Response and Production of Immunosuppressory Cytokines

Oral administration of CTB-Pins to the NOD mice led to an increasedrecruitment of immunosuppressive cytokine-producing cells (lymphocytes)to the pancreas. A large number of IL10- or IL4-producing cells are seenproximal to the pancreatic islets, which are recruited through thecirculation (FIGS. 8 & 9). This process is supported by significantperivascular migration of IL4- and IL10-expressing cells seen in thepancreas of CTBPins treated NOD mice (FIGS. 8 & 9). Blood vessels weredistinguished from ducts as follows. The internal layer lining the bloodvessels is comprised of endothelium, a thin, flat layer different fromthe more cuboidal lining of the ducts. These latter structures arethicker with a narrower lumen. In addition, endocrine glands areductless glands. Although the pancreas has both endocrine and exocrineparts, the blood vessels are more likely to be found close to theendocrine parts where the products are secreted out of the cell andabsorbed into the blood vessels, which must be in close contact to theendocrine cells. The third reason to believe that blood vessels aredepicted is that blood cells should be found in the blood vessels andnot the ducts. Also, there are no reports of immune attack against orregulatory process in favor of the pancreatic ducts.

Serum and Intestinal Immunoglobulin Levels Following Oral Delivery ofCTB-Pins

Serum and intestinal mucosal immunoglobulin (Ig) levels were determinedby ELISA using CTB as the capture antigen. Serum levels of IgG1increased in NOD mice treated with CTB-Pins expressing plant leafmaterial as compared to the control groups. There were low serum IgG2aand mucosal IgA levels against CTB observed among NOD mice treated withuntransformed plant leaf material or plants expressing CTB-IFN orCTB-GFP or CTB-Pins (FIG. 10).

Discussion

Oral administration of antigens represents a potential way to induceoral tolerance. Tolerance refers to the state of lowered systemicresponsiveness toward an antigen following oral delivery. Both activeand passive forms of tolerance can be induced, dependent upon the doseof antigen and the route of administration. The passive form is thefunctional inactivation of antigen specific lymphocytes and is selectivefor only pre-existing effectors. The active form of tolerance operatesthrough the action of regulatory lymphocytes that are able todown-modulate inflammation via bystander suppression of effector cells.Bystander suppression is viewed as a form of immunoregulation ratherthan tolerance (Homman et al., 1999b). Mucosal immunity generated byoral delivery is a protective immune response manifested by Th-2 typecytokines such as IL-4, IL 10, and TGF-[3. Antigen taken orally leads topresentation in the intestinal mucosa, which is able to generateprotective Th-2 cells in the gut associated lymphoid tissue (GALT), suchas the Peyer's patches. The antigen utilized is important because itsnature may determine the type of cytokines produced by the antigenspecific T-cells, it must direct the mucosal-derived T-cells to migrateto the organ of interest, and it must then be able to down-regulate thelocalized immune response (Gottlieb and Eisenbarth 2001). Previousstudies have demonstrated that insulin given orally to non-obesediabetic (NOD) mice reduced the level of diabetes by 50%, and theprotection was associated with the development of a Th-3-type response,specifically TGF-β producing T-cells (Zhang et al., 1991).

Oral tolerance induced by autoantigens has been applied successfully asa therapeutic tool in experimental models of autoimmune diseases(Strobel et al., 1998). The basic mechanism of oral tolerance in humansis currently a work in progress, and oral antigen administrationregimens have resulted in limited success when applied to patients(Garside et al., 1999, Pozzilli et al., 2000b, Chailous et al., 2000). Apossible explanation for the limited success could be due to the factthat the doses of the orally administered antigens to humans was too lowcompared to those we delivered to mice, considering the surface area ofthe intestinal absorptive epithelium (Pozzilli et al., 2000a). In thiscase, CTB may serve as the necessary co-factor required to overcome theinefficient presentation of insulin to the mucosal T-cells, resultingfrom the limited transport of native insulin across the epitheliallayer. In order for oral tolerance to become a realistic therapy forhuman autoimmune diseases, adjuvants that possess the ability to enhancethe tolerogenic potential of orally delivered antigens need to beidentified. The coupling of autoantigens—in this case, proinsulin—to thenon-toxic Cholera toxin B subunit (CTB) dramatically increases theirtolerogenic potential (Sun et al., 1994, Bergerot et al., 1997; Arakawaet al., 1998). This effect is mediated by the ability of CTB to act as atransmucosal carrier, although CTB may have a direct affect on theimmune system (Burkart et al., 1999, Li et al., 1996). The currentprimary limitation in advancing this concept in clinical trials is thelow levels of expression in transgenic plants (Bergerot et al., 1997).Therefore, this limitation can be overcome by the hyperexpression ofCTB-proinsulin fusion protein in transgenic chloroplasts.

Previous studies to express CTB-proinsulin fusion protein in plants wereperformed with potato plants (Arakawa et al., 1998). Expression levelsin nuclear transgenic potato plants were 0.05-0.1% total soluble protein(tsp). The low expression levels required feeding NOD mice with largeamounts of fresh potatoes. In this study, we have expressedCTB-proinsulin fusion protein in transgenic tobacco chloroplasts tolevels at least 130-300-fold greater than those in nuclear transgenicpotatoes. As such, we fed the NOD mice 8 mg of leaf tissue per week,which is a 375-fold lesser amount of plant tissue compared to the 3 gper week used previously (Arakawa et al, 1998). Using these smallconcentrated doses reduces the possibility of potential confoundingeffects of leaf tissue and eliminates the need to process or purify theplant material. Such hyperexpression of CTB-Proinsulin should make thisfusion protein abundantly available for animal studies in NOD mice.Expression of CTB-Proinsulin within transgenic chloroplasts alsoeliminates the detrimental effects that occurred to nuclear transgenicproteins in the cytoplasm (Mason et al., 1999). Secondly, the expressionof the foreign gene within chloroplasts provides a safeguard fortransgene containment due to maternal inheritance of the plastid genome(Daniell, 2002) and engineered cytoplasmic male sterility (Ruiz &Daniell, 2005). Oral delivery of biopharmaceutical proteins expressed inplant cells reduces their cost of production, purification, processing,cold storage, transportation and delivery.

Oral administration of self antigens such as insulin leads to theiruptake by the gut associated lymphoid tissue (GALT), including theintestinal mucosal M cells, which pass the antigen to underlying antigenpresenting cells, such as macrophages and dendritic cells (Limaye etal., 2006). This leads to the activation of T-cells and induction of aTh2 cell response, which is characterized by the up-regulation ofimmunosuppressive cytokines (such as IL10 and IL4) and serum antibodies(such as IgG1 but not IgG2a) (Salmound et al., 2002, Farmer and Weiner2005). No significant increase in mucosal IgA was seen in our study inCTB-Pins treated mice versus the control groups.

The presence of CTB in the intestine ensures an effectivereceptor-mediated oral delivery of intact plant-derived fusion proteinacross the intestinal mucosa via binding of CTB to the GM1 gangliosidereceptor and uptake by intestinal M cells and enterocytes. Takentogether, the data presented here suggest that the suppression of thedisease was mediated by regulatory Th-2-cells. Since T-cell regulationis a major player in mucosal immunity, oral administration of anautoantigen can be used to treat autoimmune diseases in animal models bygenerating active T-cell suppression. Mucosal autoantigen administrationrepresents a potential way to establish tolerance towards autoantigensand the prevention of autoimmune diseases. Several autoimmune diseasesand their antigens are known: Multiple Sclerosis (MBP and PLP),Arthritis (Type II collagen), Uvetis (S-antigen and IRBP), Myastheniagravis (AChR) and Thyroiditis (Thyroglobin) (Hafler and Weiner 1997).

One previous human clinical study on the oral delivery of insulin wasunsuccessful (Skyler et al., 2005) because insulin was not protectedfrom digestive enzymes and acid hydrolysis. In this study, however,insulin was protected by bioecapsulation within plant chloroplasts.Future experiments involving the CTBPro insulin construct, includinghuman clinical trials, will utilize cultured cells free of nicotine orother alkaloids instead of leaves, although the amount of nicotinepresent will be negligible. Additionally, we used 5-week old mice inthis study to demonstrate the alleviation of symptomatic pancreaticinsulitis and preservation of insulin-producing β-cells in mice, acondition that mimics human type 1 diabetes. Based on the success of theconcept in older mice (Harrison et al., 1996), this strategy is likelyto work not only prior to the onset of diabetes, but also at laterstages of this autoimmune disease.

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(2003) Phytoremediation of    organomercurial compounds via chloroplast genetic engineering. Plant    Physiol. 132: 1344-1352.-   Ruiz, O. & Daniell, H. (2005). Engineering Cytoplasmic male    sterility via. The chloroplast Genome by expression of    β-ketothiolase. Plant Physio. 138, 232-246.-   Salmond R J, Luross J A, Williams N A. (2002) Immune modulation by    the cholera-like enterotoxins. Expert Rev Mol. Med. October 1;    2002:1-16. Review.-   Samsam M, Mi W, Wessig C, Zielasek J, Toyka K V, Coleman M P,    Martini R (2003) The Wlds mutation delays robust loss of motor and    sensory axons in a genetic model for myelin-related axonopathy. J.    Neurosci. 1; 23(7):2833-9.-   Singleton M L. (2003) Expression of CaF1 and LcrV as a fusion    protein for development of a vaccine against Yersisnia pestis via    chloroplast genetic engineering. Master's Thesis, University of    Central Florida.-   Skyler J S., Krishner J P., Wolfsdorf J., Cowie C., Plamer J P.,    Greenbaum C., Cuthbertson D., Rafkin-Mervis L E., Chase H P., and    Leschek E. (2005) Effects of oral insulin in relatives of patients    with type 1 diabetes. Diabetes Care 28: 1068-1076-   Staub J M, Garcia B, Graves J, Hajdukiewicz P T, Hunter P, Nehra N,    Paradkar V, Schlittler M, Carroll J A, Spatola L, Ward D, Ye G,    Russell D A (2000) High-yield production of a human therapeutic    protein in tobacco chloroplasts. Nat. Biotechnol. 18: 333-338.-   Stern D B, Gruissem W (1987) Control of plastid gene expression: 3′    inverted repeats act as mRNA processing and stabilizing elements,    but do not terminate transcription. Cell. 51: 1145-57.-   Strobel S, Mowat A M. (1998) Immune responses to dietary antigens:    oral tolerance. Immunol Today. April; 19(4):173-81. Review.-   Sun, J-B., Holmgren J. Czerkinsky C. (1994). Cholera toxin B    subunit: an efficient transmucosal carrier-delivery system for    induction of peripheral immunological tolerance. Proc Natl Acad.    Sci. USA 91: 10795-10799.-   Tregoning J S, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F,    Fairweather N, Ytterberg J, van Wijk K J, Dougan G, Maliga P. (2003)    Expression of tetanus toxin fragment C in tobacco chloroplasts.    Nucleic Acids Res. 31: 1174-1179.-   Watson J, Koya V, Leppla S, Daniell H. (2004) Expression of Bacillus    anthracis protective antigen in transgenic chloroplasts of tobacco,    a non-food/feed crop. Vaccine 22: 4374-4384.-   Zhang Z J, Davidson L, Eisenbarth G, Weiner H L. (1991) Suppression    of diabetes in nonobese diabetic mice by oral administration of    porcine insulin. Proc Natl Acad Sci USA. November 15; 88    (22):10252-6

Example 2 Expression of a Cholera Toxin B Subunit-Rotavirus EnterotoxinFusion Gene in Transgenic Nicotiana tabacum Chloroplast Introduction

Rotavirus, the major cause of life-threatening infantilegastroenteritis, is a member of the Reoviridae family and is consideredto be the single most important cause of virus-based severe diarrhealillness in infants and young children particularly 6 months to 2 yearsof age in industrialized and developing countries. Rotaviruses belong tothe family Reoviridae and are spherical 70-nm particles. The virusgenome contains 11 segments of double-stranded RNA, each encoding aviral capsid or nonstructural protein [1]. The identification of arotavirus nonstructural protein gene (NSP4) encoding a peptide, whichfunctions both as a viral enterotoxin and as a factor involved in theacquisition of host cell membrane during virus budding from cells,provides a new approach for mucosal immunization. NSP4 has beendesignated as the viral enterotoxin as it was demonstrated that apeptide derived from its cytoplasmic domain is enough to cause Diarrheain 3-Day old mice [2]. Various critical functions of NSP4 at themolecular level have also been identified; it plays a major role inviral morphogenesis by functioning as an intracellular receptor to aidin the budding of subviral particles into the endoplasmic reticulum (ER)[3]. It has been demonstrated that NSP4 possesses membranedestabilization activity on ER by mobilizing intracellular calcium andhence increasing its levels in intestinal cells. It also affects themembrane trafficking from the ER to the Golgi complex with its abilityto bind to the micro tubules [4]. NSP4 induced intracellular calciummobilization may be responsible for some of the cellular aspects ofrotavirus pathogenesis as this increase in intracellular calciumultimately stimulates endogenous fluid secretory pathway in theintestinal mucosa [5]. These above attributes of C-terminal portion ledto the use of the truncated form of NSP4 with 90 amino acids as a goodcandidate for rotavirus vaccine antigen instead of the full length NSP4.Cholera toxin B subunit (CTB) of Vibrio cholera has been shown tofunction efficiently as an adjuvant and carrier molecule for foreignproteins and especially for mucosal vaccines. Direct linking of smallantigens with CTB results in specific targeting of the antigens to themucosal immune system through its specific binding affinity to GM1receptors of enterocytes and also increases the local antigenconcentration at the mucosal surface. Hence the immune response toCTB-NSP4 fusion protein is expected to be lot stronger⁶.

Presently there is no available vaccine for rotavirus included innational immunization systems, the only live tetravalent rhesus-humanreassortant vaccine (RRV-TV; Rotashield) for rotavirus was licensed in1998 in USA but withdrawn from market in 1999 for possible associationwith intussusception⁷. Among the various protein expression systemsavailable, genetically engineered plants are considered to be mosteconomical. As lot of investment is needed to establish and maintain theindustrial facilities using fermentation or bioreactors when compared tothe technology that is already available for harvesting and processingplants and plant products on a large scale [8, 9]. Plant-derivedproducts are less likely to be contaminated with human pathogenicmicroorganisms than those derived from animal cells because plants don'tact as hosts for human infectious agents 10. Recombinant proteinsexpressed in plant cells are naturally protected from degradation whentaken orally 11. The levels of recombinant proteins expressed intransgenic plants by nuclear system have been observed to be less than1% of total soluble protein which is considered to be commerciallyunfeasible for protein purification 8.

The inventors have realized that one of the most attractive alternativemeans for achieving higher expression levels of foreign proteins inplants is through the chloroplast transformation. Chloroplasttransformation is considered to be an ideal system for expressingforeign proteins as it offers several advantages like high-leveltransgene expression 12, proper folding of proteins, multi-geneengineering in a single transformation event 12, 13, transgenecontainment via maternal inheritance [14, 15, 16, 17], lack of genesilencing, position effect due to site-specific transgene integration.Chloroplasts also possess the ability to accumulate any foreign proteinsin large amounts that could otherwise be harmful if they were in thecytoplasm. For example CTB an oral subunit vaccine for cholera was nottoxic when expressed in transgenic plastids in very high quantitieswhich were otherwise toxic when expressed in leaves by nucleartransformation. Trehalose, a pharmaceutical industry preservative wastoxic when accumulated in cytosol where as was non toxic whencompartmentalized in plastids by chloroplast expression system^(14,18).

The various vaccine antigens and therapeutic proteins have beensuccessfully hyperexpressed via the chloroplast genetic engineering.Vaccine antigens that have already been expressed in the chloroplastinclude the Cholera toxin B-subunit (CTB) 15, the F1˜V fusion antigenfor plague 19, the 2L21 peptide from the Canine Parvovirus (CPV) 20,Anthrax Protective antigen (PA) 21, LecA protein as vaccine antigen forEntamoeba histolytica 22, NS3 protein as vaccine antigen for hepatitis C23, C terminus of Clostridium tetani (TetC) (24, 25) and therapeuticproteins like Human Serum Albumin 26, Magainin 27, Interferon andInsulin like growth factor 28.

Plastid transformation has been proven to be highly successful intobacco. The popularity of tobacco is due to the availability of welldefined regulatory elements for the transgene expression. Tobacco beinga non food, non feed crop carries a reduced risk of transgenic maternalor recombinant proteins contaminating feed and human food chains. One ofthe other advantages of tobacco crop is its ability to yield highbiomass (produces in excess of 40 metric tones of leaf fresh weight peracre on multiple harvests annually) with low maintenance andcost^(29,30). For these attractive reasons tobacco plastidtransformation has been a successful vehicle for the large scaleproduction of human recombinant proteins and vaccines too.

The expression levels of CTB-NSP4 that were achieved in transgenicpotato by nuclear expression was about 0.006% to 0.026% which is notfeasible for purification 31. Hence the main objective of this projectis to express the surface antigen CTB-NSP4₉₀ fusion gene in plants usingthe chloroplast expression system to achieve high levels of expressionto enhance the protective efficacy and also develop a low cost vaccinefor rotavirus.

Results

Construction of pLD-5′UTR-His-CTB-NSP4 Vector for Tobacco ChloroplastTransformation.

The pRSET vector with Histag-CTB-NSP4 cloned in its multiple cloningsites was a gift from Dr William H R Langridge, Loma Linda UniveristySchool of Medicine. The goal was to clone the cassette into theuniversal chloroplast transformation vector, pLD-Ctv under the controlof light regulated psbA 5′UTR regulatory sequence which enhances thetranslation of the genes. The Histag-CTB-NSP4 gene cassette in pRSETvector was digested with Nde I and EcoRI and cloned into p-bluescriptcontaining the 5′UTR regulatory sequence as shown in FIG. 11C named asp-bluescript-5′UTR-Histag-CTB-NSP4. The p-bluescript containing the5′UTR-Histag-CTB-NSP4 cassette was then digested with EcoRV and XbaI andwas cloned into the universal chloroplast transformation vector, pLD-Ctvwithin the EcoRV and XbaI sites and was designated as pLD-AK as shown inFIG. 11D. The pLD vector contains the homologous recombination sequences(flanking sequences) that allowed the homologous recombination of thegene cassette (aadA, 5″UTR-His-CTB-NSP4) in between the trnI and trnA ofthe chloroplast genome 15. Downstream to the trnI, the vector providedthe constitutive 16S rRNA promoter, which regulates the expression ofaadA gene (aminoglycoside 3′ adenyltransferase) that confers resistanceto spectinomycin-streptomycin and the 5′UTR-His-CTB-NSP4 gene encodingthe cholera toxin B subunit-rotavirus NSP4 enterotoxin fusion protein.Upstream to the trnA, the vector contains the 3′UTR which is atranscript stabilizer derived from the psbA gene.

After recovering in the dark for 48 hours from bombardment, leaves werecut into 5 mm² pieces and placed on RMOP 32 plates containing 500 μg/mlspectinomycin for Petite Havana, for the first round of selection asdescribed in Daniell 33,34,35. From 10 bombarded Petit Havana leaves,about 20 green shoots appeared after 4 weeks. For second round ofselection the leaves were cut into 2 mm² pieces and then transferred tofresh RMOP plates with 500 μg/ml spectinomycin for Petite Havana 33, 35.

The shoots that appeared during the second round of selection weretested positive for cassette integration into the chloroplast genome byPCR analysis, were grown in sterile jars containing fresh plant MSOmedium with spectinomycin until the shoots grew to fill the jars. Thenthe plants were transferred to pots with soil containing no antibiotic.Potted plants were grown in a 16 hour light/8 hour dark photoperiod inthe growth chamber at 26° C.

Transgene Integration in Chloroplast and Homoplasmy

After bombardment of tobacco leaves with gold particles coated withplasmid DNA (pLD-5′UTR-His-CTB-NSP4), about 5 shoots/plate appearedafter a period of 5-6 weeks. The shoots that were obtained on the RMOPselection medium could be due to any one of the three possible and twotypes of integration: chloroplast transgenic, nuclear transgenic ormutant shoots. Spontaneous mutation of the 16S rRNA gene, which confersresistance to spectinomycin in the ribosome, could allow plants to growon spectinomycin without integration of the gene cassette which willresult in the mutant shoot growth. The aadA gene in the gene cassetteconfers resistance to spectinomycin and hence the shoots with theintegration of the gene cassette in either nuclear or chloroplast genomegrow on the selection medium. True chloroplast transformants weredistinguished from nuclear transformants and mutants by PCR analysis.Two primers, 3P and 3M were used to test for chloroplast integration oftransgenes 15. 3P primer lands on the native chloroplast DNA in the 16SrRNA gene region and the 3M primer lands on the aadA gene as shown inFIG. 11A. Nuclear transformants were eliminated because 3P will notanneal and mutants were eliminated because 3M will not anneal. The 3Pand 3M primers upon chloroplast integration of transgene will yield aproduct of 1.65 kb size fragment as shown in FIG. 11B.

The Integration of the aadA, 5′UTR-His-CTB-NSP4 gene and 3′psbA UTR,were additionally tested by using the 5P and 2M primer pair for the PCRanalysis. The 5P and 2M primers annealed to the internal region of theaadA gene and the internal region of the trnA gene respectively as shownin FIG. 11A 15. The product size of a positive clone is of 2.5 kb forCTB-NSP4, while the mutants and the control do not show any product.FIG. 11C shows the result of the 5P/2M PCR analysis. After PCR analysisusing both primer pairs, the plants were subsequently transferredthrough different rounds of selection on spectinomycin media to obtain amature plant and reach homoplasmy.

Southern Analysis of Transgenic Plants

The plants that tested positive for the PCR analysis were moved throughthree rounds of selection and were then tested by Southern analysis forsite specific integration of the transgene and homoplasmy. The DNA ofthe full regenerated clones growing in jars (third selection) wasextracted and used for the Southern analysis. The flanking sequenceprobe of 0.81 kb in size allowed detection of the site-specificintegration of the gene cassette into the chloroplast genome; this wasobtained by double digesting the pUC-Ct vector that contained the trnIand trnA flanking sequences (FIG. 12A) with BamHI and BglII 15. FIG. 12Bshows the HincII sites used for the restriction digestion of the plantDNA for pLD-5′UTR-Histag-CTB-NSP4. The transformed chloroplast genomedigested with HincII produced fragments of 4.3 kb and 2.0 kb forpLD-5′UTR-Histag-CTB-NSP4, while the untransformed chloroplast genomethat had been digested with HincII resulted in a 5.0 kb fragment (FIG.12D). The flanking sequence probe can also show if homoplasmy of thechloroplast genome has been achieved through the three rounds ofselection. The plants expressing CTB-NSP4 showed homoplasmy as there isno wild type band seen in transgenic lines within the levels ofdetection. The gene specific probe CTB-NSP4 of size approx. 0.7 kb wasused to show the specific gene integration producing a fragment of 11 kbwhen CTB-NSP4 transgenic plant DNA was digested with ClaI as shown inFIGS. 12C and 12E.

Immunoblot Analysis

Crude protein extract of 20 ug, was loaded in each well of the SDS-PAGE.The rabbit anti-NSP4₉₀ antibodies (provided by Dr. William Langridge,Loma Linda Univ. of Loma Linda) were used to detect the 27 kDa and 135kDa monomeric and pentameric forms CTB-NSP4 fusion protein (FIG. 13).The wild type plant (Petit havana) did not show any bands indicatingthat the anti-NSP4 antibodies did not cross react with any otherproteins in the crude extract. As the CTB-NSP4 expression level was2.45% of TSP in mature leaves, which indicates that there is 2.4 ug ofthe fusion protein in 100 ug of TSP. the total crude protein extractloaded in each well is 20 ug and so the expected amount of CTB-NSP4fusion protein present in 20 ug of TSP will be about 0.6 ug. Hence eachof the wells contains approximately about 0.6 ug of the CTB-NSP4 proteindetected by the CTB-NSP4 antibodies.

Protein Quantification and Binding Affinity Using GMI Binding AssayELISA

The levels of pentameric CTB-NSP4 fusion protein in transformed tobaccoplants and its affinity for GM1-ganglioside was evaluated byquantitative GM1 ELISA. The standard curve has been obtained usingdifferent dilutions of purified CTB-NSP4. The dilutions were made in0.01M phosphate buffered saline (PBS). The primary antibody used wasRabbit antibody raised against NSP4₉₀ protein expressed and purifiedfrom Ecoli BL21 cells and secondary antibodies were donkey anti-rabbitantibodies peroxidase conjugated. The percentage of CTB-NSP4 expressedwas as a percent of total soluble protein calculated using the Bradfordassay i.e. the percentage of CTB-NSP4 is inversely proportional to theTSP values. The CTB-NSP4 expression levels reached a maximum of 2.45% ofthe total soluble protein in the mature leaves after 1 Day of continuouslight exposure due to increase in translation obtained under the controlof light regulated psbA 5,UTR as shown in FIG. 14. The increasedexpression in mature leaves is due to more number of chloroplasts andhigh number of chloroplast genomes (up to 10,000 copies/cell) in themature leaves. Also, the large size and more number of mature leaves perplant contributed to the higher levels of CTB-NSP4 in mature leaves.

Discussion

The pLD-5′UTR-Histag-CTB-NSP4 chloroplast transformation vectorcontaining the aadA gene, CTB-NSP4 coding region and 3′ psbA, integratesthe transgene cassette into the transcriptionally active trnI-trnAspacer region of the chloroplast genome via homologous recombination.The site directed insertion of CTB-NSP4 into the chloroplast genome isachieved by homologous recombination between trnI-trnA regions ofpLD-5′UTR-His-CTB-NSP4 and plastid genome which prevents any randomintegration of transgene that is usually observed with nucleartransformation. Achieving high expression of the CTB-NSP4 recombinantfusion protein in the chloroplast depends on various factors. First, thepLD-His-CTB-NSP4 vector is designed to integrate into the invertedrepeat region of the chloroplast genome via homologous recombination.When the CTB-NSP4 fusion gene is inserted into the IR region, the copynumber of the transgene gets doubled by a phenomenon know as copycorrection that recruits the introduced transgene into another IR region36,37). Increased copy number results in increased transcript levelsresulting in higher protein accumulation 35, 12. Second, the psbA 5′ UTRtypically has stem loop structure which aid in transcript stability andis also a binding site for translation activation factors to enhance thebinding of ribosomes to the mRNA for efficient translation. Also thetranslation of psbA mRNA is stimulated by light proposed to be mediatedby a nuclear encoded protein. The binding of the nuclear encoded (RB)protein and psbA is directly dependent light which there by enhances theinitiation of translation. The redox potential generated by lightreactions of photosynthesis is used by chloroplast Protein DisulfideIsomerase system and thioredoxin which then activate the binding oftranslation activation factors to the ribosome binding sites in psbA 5′UTR thereby enhancing the translation in the presence of light 38. Theexpression of CTB-NSP4 in transgenic plant under continuous light showedan increase in expression at Day 1. The psbA 3′ untranslated region(UTR) used for the regulation of transgene expression has potential rolein post transcriptional stabilization by binding to different RNAbinding proteins and help in enhancing translation of the foreignprotein 14. Third, the pLD-His-CTB-NSP4 vector consists of a OriA sitefor origin of replication within trnI flanking region allowing to attainhomoplasmy even in the first round of replication by increasing thenumber of templates for integration into the chloroplast genome (36, 37.To obtain an optimal production of the CTB-NSP4 fusion protein andtransgene stability, it is essential to achieve homoplasmy throughseveral rounds of selection on media containing spectinomycin. Ifhomoplasmy is not achieved, it could result in heteroplasmy which leadsto changes in the relative ratios of the two genomes upon cell division.The presence of heteroplasmic condition in a transgenic plant mightretrograde back to the wild type eliminating the transgene in theabsence of selection pressure in subsequent generations. The chimeric,aminoglycoside 3′ adenyl transferase (aadA) gene, conferring resistanceto spectinomycin was used as a selectable marker and its expression isdriven by the 16S (Prrn) promoter (15, 39). Spectinomycin binds the 70Sribosome and inhibits translocation of peptidal tRNA's from the A siteto the P site during protein synthesis. The aadA gene codes for theenzyme aminoglycoside 3′ adenlyltransferase, which transfers the adenlylmoiety of ATP to spectinomycin and inactivating it. Fourth, chloroplasttranslation system provides the necessary enzymes for proper folding anddisulphide bond formation. Chaperonins present in chloroplast arethought to aid in the folding and assembly of non native prokaryotic andeukaryotic proteins (15, 40). Reversible activation of genes thatregulate expression in the chloroplast is the Protein DisulfideIsomerase (PDI) system composed of chloroplast polyadenylate-bindingproteins that specifically bind to the 5′UTR of the psbA mRNA and aremodulated by redox status through PDI (37). The ability of chloroplaststo form disulfide bonds and properly fold foreign proteins eliminates amajor part of the costly downstream processing. The chloroplastexpressed CTB-NSP4 fusion protein folded properly into functionalpentameric form which was clearly seen on the immunoblot. The positiveresult in GM1 binding assay with CTB-NSP4 has reconfirmed the pentamerforms of CTB-NSP4 from transgenic tobacco chloroplasts.

Chloroplast transformants were distinguished from the nucleartransformants and mutants by PCR analysis. Southern blot analysis withgene specific CTB-NSP4 probe and flaking probe for chloroplast genomewas done to confirm the site-specific integration of the gene cassetteand also to determine the homo or heteroplasmy. High protein expressionlevels were obtained in the mature leaves after Day 1 of continuouslight exposure of up to 2.45% of the total soluble protein which wasquantified using the GM1 binding assay.

The present study reports the successful expression of the CTB-NSP4fusion protein as pentameric forms. This opens the doors for theexpression of CTB-NSP4 in carrot plastids so as to enable oral deliveryof the vaccine antigen. The immunogenecity of the vaccine antigen needsto be tested in an animal model which is underway.

Materials and Methods

Construction of pLD-5′UTR-HisCTBNSP4 Vector for Transformation ofTobacco Chloroplast

Initially the gene cassette Histag-CTB-NSP4 was cloned downstream to5′UTR in p-bluescript between EcoRV and EcoRI sites. Then the final genecassette containing the 5′UTR and His-CTB-NSP4 (approximate size 0.7 kb)were digested with EcoRV and XbaI and cloned into tobacco universalvector pLD-Ctv between EcoRV and XbaI.

Bombardment and Transgenic Plant Regeneration

Sterile Nicotiana tabacum cv. Petit Havana tobacco leaves were bombardedusing the Bio-Rad PDS-1000/He biolistic device as previously described[32,33,35]. The bombarded leaves were allowed to incubate in dark for 48hours to recover from tissue damage and then were placed on RMOP mediumcontaining 500 ng/ml spectinomycin for two rounds of selection on platesand subsequently moved to jars with MSO medium containing 500 ng/mlspectinomycin³⁵.

Confirmation of Transgene Integration into the Chloroplast Genome

To confirm the transgene cassette integration into the chloroplastgenome, PCR was performed using the primer pairs 3P(5′-AAAACCCGTCCTCGTTCGGATTGC-3′)-3M(5′-CCGCGTTGTTTCATCAAGCCTTACG-3′) 15and the complete transgene integration was confirmed by PCR analysisusing primer pairs 5P (5′-CTGTAGAAGTCACCATTGTTGTGC-3′) and 2M(5′-GACTGCCCACCTGAGAGC-GGACA-3′) 15. The total DNA from putativetransgenic and untransformed tobacco plants was isolated using QiagenDNeasy Plant Mini Kit. The PCR reaction was set as follows: 150 ng ofplant DNA, 1× Taq buffer, 0.5 mM dNTPs, 0.2 mM of primers each, 0.05units/μl Taq polymerase. The amplification was set for 30 cycles with aprogram timed in the following way: 94° C. for 30 sec, 65° C. for 30sec, and 72° C. for 30 sec for the 3P-3M primer pair and 72° C. for 1min for the 5P-2M primer pair. Cycles were preceded by denaturation for5 min at 94° C. and followed by a final extension for 7 min at 72° C.PCR products including the controls were loaded into a 0.8% agarose gelto confirm the results.

Southern Blot Analysis

The total plant DNA was digested with HincII and probed with Chloroplastflanking probe. The total plant DNA was also digested with ClaI in thesimilar manner and probed with CTB-NSP4 gene specific probe. The aboveset of digested samples was run on 0.7% agarose gel. The gels weresoaked in 0.25 N HCl for 15 minutes and then rinsed 2 times with water.The gels were later soaked in transfer buffer (0.4 N NaOH, 1 M NaCl) for20 minutes and then transferred overnight to a nitrocellulose membrane.The membranes were rinsed twice in 2×SSC (0.3 M NaCl, 0.03 M Sodiumcitrate), dried on filter paper, and then crosslinked in the GSGeneLinker (Stratagene, La Jolla, Calif.)³⁵. The flanking sequence probewas made by digesting pUC-CT vector DNA with BamHI and BglII to generatea 0.81 kb probe lee et al. The CTB-NSP4 sequence of about 0.7 kb wasused as gene specific probe. The probes were labeled with 32P using theProbeQuant G-50 Micro Columns (Amersham, Arlington Heights, Ill.). Theprobes were hybridized with the membranes using Stratagene QUICK-HYBhybridization solution and protocol (Stratagene, La Jolla, Calif.).

Immunoblot Analysis

To detect the CTB-NSP4 fusion protein expression in transgenic tobaccoplants the total protein was extracted from 100 mg of leaf tissue in 200μl of plant extraction bufer (0.1% SDS, 100 mM NaCl, 200 mM Tris-HCl pH8.0, 0.05% Tween 20, 400 mM sucrose, 2 mM PMSF). Similarly total proteinfrom 100 mg of untransformed tobacco plant was also extracted to use ascontrol. Both boiled (4 minutes) and unboiled samples of extractedprotein with sample loading buffer were separated on 10% SDS/PAGE gelfor one hour at 50V and then 3-4 hours at 80V for uniform separation.The proteins thus separated were transferred to a nitrocellulosemembrane by electroblotting at 85V for one hour. The membrane wasinitially blocked with PTM (1×PBS, 0.05% Tween 20, and 3% dry milk) forone hour. Followed by incubation in P-T-M containing diluted (1:3000)rabbit anti-NSP4₉₀ antibody (provided by Dr Langridge, Univ of LomaLinda). Membranes were then washed with distilled water and transferredto P-T-M containing diluted (1:5000) goat derived anti-rabbit IgGantibody conjugated with alkaline phosphatase (AP) (Sigma, nationalimmunization systems St. Louis, Mo.). Blots were washed three times withPBST for 15 minutes each time. Then washed with PBS for 10 minutes,followed by addition of chemiluminiscent substrate ((Pierce, Rockford,Ill.) for AP and incubating at room temp for 5 min for thechemiluminescence. Later the X-ray films were exposed tochemiluminescence and the films were developed in the film processor tovisualize the bands.

Bradford Assay for Protein Quantification (Bio-Rad Manual).

The Bradford assay was used to determine the total protein from theplant extracts prepared as described above. This was used to determinethe percent of CTB-NSP4 antigen in the total soluble protein extract (or% TSP). An aliquot of plant extract as prepared above was thawed on ice.Extraction buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, 0.2 g NaN3, 0.1% Tween20, and 5 mM PMSF adjusted to pH 9.6) was used to make Bovine SerumAlbumin (BSA) standards ranging from 0.05 to 0.5 Plant extracts werediluted 1:10 and 1:20 with extraction buffer. 10 μl of each standard and10 μl of each plant dilution was added to the wells of a 96 wellmicrotiter plate (Cellstar) in duplicates. Bradford reagent (Bioradprotein assay) was diluted 1:4 with distilled water as specified and 200μl was added to each well. Absorbance was read. Comparison of theabsorbance to known amounts of BSA to that of the samples was used toestimate the amount of total protein.

GM1 Binding (ELISA) Assay

The quantification and binding affinity of chloroplast derived CTB-NSP4for GM1-ganglioside receptor in the plant crude extract was done usingthe GM1 ganglioside binding affinity (ELISA) as described by⁴¹. 100 mgof transgenic leaf samples (young, mature, old) and the wild type leafsamples (young, mature, old) were collected. The leaf samples werecollected from plants exposed to regular lighting pattern (16 h lightand 8 h dark), 1 Day, 3 Day and 5 Day continuous light exposure. Theleaf samples were finely ground in liquid nitrogen, followed bycollection of leaf powder into the eppendorf tube. Total soluble proteinfrom the plant leaves was extracted in plant protein extraction buffer(15 mM Na₂CO₃, 35 mM NaHCO₃, 3 mM NaN₃, pH 9.6, 0.1% Tween, and 5 mMPMSF). The microtiter plate was coated initially with (100 μl/well) withmonoganglioside-GM1 (Sigma) (3.0 μg/ml in bicarbonate buffer pH 9.6) andincubated overnight at 4° C. followed by washing three times with PBST(PBS and 0.05% Tween 20) and two times with dH₂0. As control, BSA (3.0μg/ml in bicarbonate buffer pH 9.6) was coated in some wells. The wellswere then blocked with 1% BSA in 0.01M phosphate buffer saline (PBS)(300 μl/well) for 2 h at 37° C. or incubate overnight at 4° C. followedby 3 washes with PBST and 2 washes with dH₂O. In order to check theprotein concentration, the standards, test samples and antibody werediluted in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, 3 mM NaN₃, pH9.6). The standards and protein samples (100 μl) were coated to 96-wellpolyvinyl chloride microtiter plate (Genstar) for 1 h at 37° C. orincubate overnight 4° C. followed by 3 washes with PBST and 2 washeswith water. The primary rabbit anti-NSP4 antibody (provided by Dr.Langridge, Univ. of Loma Linda) diluted (1:1500) in 0.5% BSA in 1×PBSwas loaded into wells and incubated for 2 h at 37° C. followed bywashing steps and then again incubated with 100 μl of donkey anti-rabbitIgG-HRP conjugated antibody made in goat (American Qualex) (1:3000)diluted in 0.5% BSA in 1×PBS. The plate was then incubated for 2 h at37° C. After the incubation the plate was washed thrice with PBST andtwice with water. The wells were then loaded with 200 μl of3,3,5,5-tetramethyl benzidine (TMB from American Qualex) substrate andincubated for 10-15 min at room temperature. The reaction was terminatedby adding 50 μl of 2N sulfuric acid per well and the plate was read on aplate reader (Dynex Technologies) at 450 nm. (Modified form of protocolfrom Ausubel et al., 4^(th) edition).

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Example 3 Expression of Hepatitis C Virus Non Structural 3 Antigen inTransgenic Chloroplasts

Hepatitis C virus infection is the major cause of acute hepatitis andchronic liver disease. An estimated 180 million people are infectedglobally (WHO). There is no vaccine available to prevent hepatitis C andtreatment with antiviral drugs is expensive and is accompanied withvarious side effects. Therefore, there is an urgent need for thedevelopment of effective vaccine antigens and an efficacious HCVvaccine. The non-structural 3 protein of the hepatitis C virus is one ofthe most conserved and multifunctional protein of the virus andtherefore is a good candidate for the development a HCV vaccine. Vaccineantigen production via chloroplast transformation system usually resultsin high expression levels and eliminates the possibility ofcontamination with viral vector sequences, human or animal pathogens. Toexpress the HCV NS3 antigen in the chloroplast of Nicotiana tabacum var.Petit havana and LAMD-609, the NS3 gene (1.9 kb) was cloned into achloroplast expression vector, pLD-ctv containing the 16S rRNA promoter,aadA gene coding for the spectinomycin selectable marker, psbA 5′ & 3′untranslated regions to enhance translation in the light and trnI & trnAhomologous flanking sequences for site specific integration into thechloroplast genome. Chloroplast integration of the NS3 gene was firstconfirmed by PCR. Southern blot analysis further confirmed site-specificgene integration and homoplasmy. The NS3 protein was detected intransgenic chloroplasts by Immunoblot analysis. The NS3 protein wasfurther quantified by ELISA. Maximum expression levels of NS3 up to 2%in the total soluble protein were observed even in old leaves, upon3-day continuous illumination. These results demonstrate successfulexpression of the HCV non-structural 3 antigen in transgenic tobaccochloroplasts.

Materials and Methods

N53-pcDNA3.1 Vector.

The plasmid encoding HCV NS3 protein (initial concentration of theplasmid-280 ng/ul in H2O) was sent cloned in the commercial plasmidpcDNA3.1/V5/His-TOPO (Invitrogen). A PCR product encoding NS3 had beeninserted in this plasmid (in the cloning box) as described by theprotocol supplied by the manufacturer. The plasmid encoded NS3 with aATG and a Kozak sequence (5′ end) and a TGA (3′ end). The plasmid wastransformed into Ultra competent XL1 Blue MRF′ Tetracycline (tet) E.coli cells (Stratagene) that were endonuclease negative.

Preparation of Ultra Competent Cells (Rubidium Chloride Method).

The ultra competent cells were prepared using rubidium chloride method(http://www.nwfsc.noaa.gov/protocols-/rbcl.html). XL1 Blue MRF′ (tet) E.coli cells (Stratagene) were made competent by the rubidium chloridemethod. The E. coli glycerol stock was streaked on the LB agar plate (1liter LB broth, 15 grams agar), containing 12.5 ug/ml Tetracycline andincubated at 37° C. overnight. An isolated colony was picked and it wasgrown in 5 ml of Psi broth (per liter-5 g Bacto yeast extract, 20 gBacto Tryptone, 5 g magnesium sulfate, pH 7.6) with 5 ug/ml Tetracyclineand incubated at 37° C. for 12-16 hrs in a shaker at 225 rpm. From theovernight culture, 1 ml was taken and inoculated in 100 ml of Psi brothand was incubated at 37° C. for about 2 hours in a shaker at 225 rpm.After 2 hours, the O.D was checked at 550 nm and rechecked again afterintervals, until the O.D reached 0.48. The culture was then kept on icefor 15 minutes and then the cells were centrifuged at 3000 g/5000 rpmfor 5 minutes in a sorvall centrifuge. The supernatant was discarded andthe pellet was resuspended in 40 ml cold TFB-I solution (per 200ml-0.558 g Potassium acetate, 2.42 g rubidium chloride, 0.294 g calciumchloride, 2.0 g manganese chloride, 30 ml glycerol, pH 5.8). The cellswere centrifuged at 3000 g/5000 rpm for 5 minutes. The supernatant wasdiscarded and the cells were resuspended in 4 ml of TFB-II solution (per100 ml-0.21 g MOPS, 1.1 g calcium chloride, 0.121 g rubidium chloride,15 ml glycerol, pH 6.5) and then kept on ice for 15 minutes. Thesuspension was aliquoted (100 ul) and quick freezed in dry ice/liquidnitrogen and the aliquots were stored at −80° C.

Transformation of pcDNA3.1 Plasmid into Competent XL1 Blue MRF′ (tet) E.coli Cells.

The competent cells were removed out of −80° C. and thawed on ice. 100μl of competent cells were taken and 1 ul (100 ng) of plasmid pcDNA3.1DNA was added and mixed by gently tapping. The cells were left on icefor 30 minutes, and the tube was gently tapped every 15 minutes. Thecells were heat shocked at 42° C. for 90-120 seconds and then left onice for 2 minutes, and then 900 μl LB broth was added to the cells andthe cells were incubated at 37° C. at 225 rpm in a shaker for 45minutes. The cells were pelleted by centrifugation at 13,000 rpm for 30seconds and the supernatant was discarded. Almost 800 ul of supernatantwas discarded and only approximately 100 ul was left. The remaining 100ul of the cells were mixed well with the pellet. About 50 ul and 100 ulof the transformed cells and untransformed (control) were plated ontoLB/amp agar plates (1 liter LB broth, 15 gr agar, 100 μg/ml ampicillin,pH 7) under the hood. Plates were covered and incubated 0/N at 37° C.(http://www.nwfsc.noaa.-gov/protocols-/rbcl.html).

Rapid Colony Screening by Cracking Method.

To check the colonies for the presence of plasmids, the Rapid Screenprocedure by Promega was used. Sterile toothpicks were used for picking8 colonies from the incubated LB agar plates to the bottom of anindividual sterile microcentrifuge tube. 25 μl of 10 mM ethylene diaminetetra-acetic acid (EDTA), pH 8 was added to the tubes and vortexed tomix. Then 25 μl of fresh 2× cracking buffer (2N NaOH, 10% sodium dodecylsulfate, 1M sucrose) was added to each colony and vortexed. The tubeswere then incubated at 65° C. for 10 minutes and were cooled at roomtemperature. 1.5 μl of 4M KCl and 3.5 μl of 6× bromophenol blue (0.25%Bromophenol blue, 40% sucrose) was added to the tubes. The tubes wereplaced on ice for 5 minutes and centrifuged at 12,000 rpm for 3 minutesat room temperature. 20 μl of the supernatant form each tube was run on0.8% agarose gel to visualize which of the selected colonies containedplasmids. Positive colonies were inoculated into 5 ml of fresh LB brothwith 100 μg/ml amp and incubated overnight at 37° C. on shaker.

Midi-Prep of pcDNA3.1.

Inoculated a colony obtained from the plate in 50 ml of liquid LB broth,to which 50 μl of ampicillin (stock concentration; 100 mg/ml) was addedand incubated at 37° C. for 12 hours in a shaker. 40 ml of the overnightculture was transferred to a clean 50 ml round bottom sorvall centrifugetube. The cells were centrifuged for 10 minutes at 8000 rpm at 40 C. Thesupernatant was discarded. The pellet was resuspended by vortexing in 5ml of Solution 1 (50 mM Glucose, 10 mM EDTA, 25 mM Tris, pH-8) with 5 ulof 100 mg/ml of RNAse freshly added to it. Solution II (500 ul of 2NNaOH, 100 ul of 10% SDS, 4400 ul of sterile water) which is a cell lysissolution was prepared freshly, added, and mixed by gently inverting thetube 6-8 times and the solution turned from milky to clear. Then 5 ml ofSol III (60 ml of 5M Potassium Acetate, 11.5 M glacial acetic acid, and28.5 ml sterile dH2O) which is neutralizing solution was added to theclear solution and mixed well by inverting the tube 6-8 times and thesolution precipitated. The solution was centrifuged for 15 minutes at12,500 rpm at 40 C. The clear supernatant was poured into a new 50 mlSorvall centrifuge tube. Cold absolute ethanol (24 ml) was added to thesupernatant and mixed well by inverting the tube 6-8 times. The tube wasthen centrifuged for 10 minutes at 10,000 rpm at 40 C to pellet theplasmids. The supernatant containing contaminants was discarded. Thepellet was washed with 12 ml of 70% ethanol and resuspended by shaking.The solution was centrifuged for 5 minutes at 10,000 rpm at 40 C. Thesupernatant was discarded and the pellet was dried in a speed vacuum orair-dried before resuspending the DNA pellet in 500 ul of TE (TE: 1MTris, pH 8.0, 0.5M EDTA). The DNA sample was loaded in a 0.8% agarosegel and run at 60 volts for 30 minutes to check for plasmid isolation(Sambrook et al., 1989).

Phenol: Chloroform Extraction.

Plasmid DNA (500 ul) was taken and 250 ul Phenol and 250 ul Chloroformwas added (1:1) and mixed well. The tube was then centrifuged at 14,000rpm at 40 C for about 10 minutes. The supernatant was transferred to anew tube and 500 ul of Chloroform: IAA (Isoamyl alcohol) was added andcentrifuged at 14,000 rpm at 40 C for 10 minutes. The supernatant wastransferred to a new tube and 0.1 volume of 3M sodium acetate (pH: 5.2)was added. Absolute ethanol (900 ul) was added and mixed well byinverting several times and then centrifuged at 14,000 rpm, 40 C for 10minutes. The supernatant was discarded and the pellet was rinsed with70% ethanol (400 ul) and centrifuged for 10 minutes at 14,000 rpm at 40C. The supernatant was discarded again and the pellet was dried in aspeed vacuum or air-dried before resuspending the DNA pellet in Elutionbuffer, 10 mM Tris Cl, (Sambrook et al., 1989).

PCR Amplification of NS3 Gene.

The NS3 gene (first 134 bp) were amplified to introduce the Sac1 andSnaB1 restriction sites at the 5′ terminal end and Not1 at the 3′ end ofthe 134 bp of the NS3 gene for further subcloning. This was done toclone the 134 bp of the NS3 gene first into p-bluescript between Not1and Sac1 sites. The primers used for amplification were the NS3-F primer(5′CAGTGTGGAGCTCTTGTACGTACCACCATGGCG3′) and the NS3-R primer(5′TGGAGAGCACCTGCGGCCGCCCATCGACCTGG3′). Primers (Invitrogen) werediluted with EB to give a 100 μM stock that was stored at −20° C. ThePCR reaction was set up with 0.5 ul plasmid DNA (60 ng), 10×PCR buffer,5.0 μl of 10 mM dNTP's, 1 μl of forward primer (NS3-F), 1 μl of reverseprimer (NS3-R), 0.5 μl of Pfu polymerase and 37.0 μl of distilled,autoclaved H20 to a total volume of 50 μl.). Samples were carriedthrough 35 cycles using the following temperatures and times: 94° C. for5 minutes, 94° C. for 45 seconds, 56° C. for 45 seconds, 72° C. for 45seconds, and followed by a 10-minute extension time at 72° C. The finalPCR product (0.1 ul) was run on a 0.8% agarose gel to analyze the PCRproducts. The PCR product was purified using the PCR purification kit(Qiagen).

Ligation of the PCR Product (Sac1/SnaBI/NS3/Not1) into p-BluescriptVector.

The PCR product was ligated into p-Bluescript cloning vector(Invitrogen) between NotI and SacI restriction sites. The ligationmixture consisted of 4 μl of PCR product after PCR purification, 16 ulof p-Bluescript, 0.5 ul of T4 DNA ligase, 6.0 ul of Ligase buffer, and3.5 ul distilled, autoclaved H20 to a total of 30 ul total volume. Thesolution was gently mixed and incubated overnight at 12° C. Competent E.coli cells were taken from −80° C. freezer and thawed on ice andtransformation was started immediately after cells thawed. 15 ul of theligation mixture was mixed into a vial containing the 100 ul of E. colicompetent cells and transformation was done as previously described(Sambrook et al., 1989).

Selection of Transformants.

The p-Bluescript cloning vector has the β-galactosidase gene (lacZ).Within this coding region is a multicloning site. Insertion of afragment of foreign DNA into the multicloning site of p-Bluescriptalmost invariably results in production of an amino-terminal fragmentthat is not capable of α-complementation. Selective plates were madewith LB agar with 100 ng/ml ampicillin and 12.5 μg/ml tetracycline.About 1 hour before transformation was complete, 40 μg/ml of5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) was spread onto thetop of the plates while under the hood. X-gal is a lactose analog thatturns dark blue when it is hydrolyzed by β-galactosidase. After theX-gal dried (about 15 minutes), 40 μl of 100 mM ofisopropyl-β-D-thiogalactoside (IPTG) was spread onto the plates. IPTG,another lactose analog, is a strong inducer of lacZ transcription but isnot hydrolyzed by β-galactosidase. The plates were warmed 37° C. for 30minutes and then the plates were streaked with 100 μl of the transformedbacterial cells were spread over the top of the agar. Allowed the platesto dry for 5 minutes, and then incubated the plates in an invertedposition at 37° C. overnight. Colonies without an interrupting insertwere blue because they had an active β-galactosidase. Colonies with aninsert were white, so these were picked to culture, and midi-prep wasdone with the Midi-prep kit (Qiagen).

Sequencing of NS3 in p-Bluescript.

The PCR product in the plasmid (NS3-p-Bluescript) was sequenced usingM13 forward (5′-TGACCGGCAGCAAAATG-3′) and M13 reverse(5′GGAAACAGCTATGACC-ATG-3′) primers. Sequencing results confirmed thatthe fragment in the p-Bluescript vector was the NS3 gene.

Construction for pLD-AB-NS3 Vector for Transformation of TobaccoChloroplasts.

The original vector pcDNA3.1 was digested with BstXI and EcoRV and theNS3 gene (remaining 1760 bp) was ligated between BstXI and EcoRV inp-Bluescript. The entire NS3 gene was digested from p-bluescript withSnaBI and HindIII and ligated in pCR2.1 vector downstream of the psbA5′UTR. Finally, the pCR 2.1 vector containing the 5′UTR and the NS3 genewas digested with EcoRI and EcoRV (fragment size 2.1) and was clonedbetween the same sites in the universal vector pLD-AB-Ct.

Extraction of NS3 Protein from Transformed E. coli Cells.

5 ml of Terrific Broth (TB) containing 5 ul ampicillin (100 μg/nl) andtetracycline (50 μg/nl) was inoculated with the scrapping from theglycerol stock of E. coli transformed with pLD-AB-NS3 and incubated in ashaker at 37° C. for 10-12 hours. 5 ml of Terrific Broth (TB) withuntransformed E. coli cells was used as a negative control. The buffersand gels used in this study were made from protocols in SDS-PAGE BufferSystem (Laemmli, 1970). 800 μl of cultured cells were taken andcentrifuged for 2 minutes at 12,000 rpm. The supernatant was discardedfrom pelleted E. coli cells and then washed with 1 ml of 1×Phosphate-Buffered Saline (PBS: 140 mM NaCl, 2.7Mm KCl, 4 mM Na2HPO4,1.8 mM KH2PO4, pH 7.2). The pellet was resuspended and then centrifugedfor 1 minute at 13,000 rpm. The supernatant was then discarded. 50 μl of1×PBS was added and mixed well. 50 μl of 2× loading buffer, also calledSample Buffer or SDS Reducing Buffer (1.25 ml of 0.5 M Tris-HCl, pH 6.8,2.0 ml of 10% (w/v) SDS, 0.2 ml of 0.5% (w/v) bromophenol blue, 2.5 mlof glycerol, dH20 to a total volume of 9.5 ml, then add 50 μl ofβ-mercaptoethanol to the 9.5 ml) was added. The sample extracts wereboiled for 4 minutes and then immediately loaded onto gels (Sambrook etal., 1989).

SDS-PAGE.

The buffers and gels used in this study were made from protocols inSDS-PAGE Buffer System below (Laemmli 1970). To detect the proteinextracted from E. coli cells containing pLD-AB-NS3, SDS-PAGE gels weremade in duplicate utilizing the following solutions: 1.) Bio-Rad(cat#161-0158), which is a 30% Acrylamide/Bis solution according to theratio 37:5:1. 2.) The resolving buffer, which was used to make the lowerportion of the gel: 1.5M Tris-HCl, pH 8.8. The pH was adjusted with 6NHCl and brought to a total volume of 150 ml with dH20. 3.) The stackingbuffer that was used to make the stacking gel layered over the resolvinggel and concentrated the samples at top of the resolving gel to improveresolution: 0.5M Tris-HCl, pH 6.8. 4.) Electrode buffer (1×) which wasthe gel running buffer. For 10× Electrode buffer: Dissolved 30.3 g Trisbase, 144.0 g glycine and 10.0 g SDS into 1000 ml dH20. 5.) 2× loadingbuffer also called the Sample buffer and the SDS Reducing Buffer. 6.)10% (w/v) Sodium Dodecyl Sulfate (SDS): 7.)N,N,N,N′-Tetra-methyl-ethylene diamine (TEMED) from BIO-RAD(cat#161-0800). 8.) 20% Ammonium Persulfate (APS): Dissolved 20 mg ofAPS into 1 ml dH20 and this solution can be stored at 4° C. for about amonth. To make the 10% resolving gel, in 4.1 ml dH20, 3.3 ml of 30%Acrylamide/Bis, 2.5 ml of resolving buffer and 100 μl of 10% SDS wasadded. 40 μl of 20% APS and then 10 μl of TEMED was added to the gelmixture. The gel mixture was poured between the two, vertical, glassplates leaving about 1.5 cm at the top of glass plates for the stackinggel. The gel was allowed to polymerize for 20 minutes. To make the 4%stacking gel, in 6.1 ml dH20, 1.3 ml of 30% Acrylamide/Bis, 2.5 ml ofthe stacking buffer and 100 μl of 10% SDS was added. 40 μl of 20% APSand then 10 μl of TEMED was added to the gel mixture. The 4% gel mixturewas layered on top of resolving gel and then the comb is inserted forthe formation of wells. After polymerization for about 20 minutes, thecomb is removed and put vertically into PAGE apparatus containing 1×Electrode (running) buffer. 20 μl of protein extract from pLD-AB-NS3transformed and untransformed E. coli cells was loaded along with 10 ulprotein marker. Gel was ran at 50V until samples stacked onto the top ofthe resolving gel, then ran gel at 80V for 2-3 hours so that proteinmarker bands could spread out sufficiently (Sambrook et al. 1989).

Transfer to Membranes and Immunoblot Analysis.

The separated proteins were transferred onto a 0.2 μm Trans-Blotnitrocellulose membrane (Bio-Rad) by electroblotting in Mini-TransferBlot Module at 80V for 45 minutes in Transfer buffer (360 ml of 10×Electrode buffer, 360 ml of methanol, 0.18 grams of SDS, 1080 mldistilled H20). The membranes were taken out and rinsed with water andplaced in blocking solution (100 ml 1×PBS, 100 μl of Tween 20, 5 g ofnon-fat, Carnation powdered milk) and incubated for an hour at roomtemperature in a shaker. The P-T-M was poured off and the Hepatitis CVirus (N53)-specific primary mouse monoclonal antibody (HCV NS3 Ab-1,Clone MMM33, from Neomarkers) was added in the ratio of antibody: PTM as1:1000 and incubated for 2 hours at room temperature in a shaker.Membranes were then washed with distilled water and transferred to P-T-Mcontaining goat derived anti-mouse IgG antibody conjugated withHorseradish peroxidase (Sigma, St. Louis, Mo.), in the ratio ofantibody: PTM as 1:10,000 and incubated for 1.5 hours at roomtemperature in shaker. Blots were washed three times with PBST for 15minutes each time and then washed with only PBS for 10 minutes. Then 750μl of 2× Stable Peroxidase Solution and 750 μl of 2× Luminol/EnhancerSolution (Pierce) was poured over the membrane and a film was developedin the to visualize the bands (Sambrook et al. 1989).

Sterilization of Seeds for Wild-type and T1.

For generating wild-type (untransformed) tobacco plants to use forbombardment, pods were picked from both varieties of tobacco when thepods were dry. The pods were broken under hood and then poured intolabeled eppendorf about until about ⅓ full. To germinate seeds, freshMSO (Murashige and Skoog, 1962) plates with no antibiotic were made. Thesterilization solution consisted of 1.5% bleach (4 ml of 5.25% Chloroxbleach), 16 ml d/aH20, 0.05% Tween 20 (20 μl of Tween 20). 1.2 ml of thesterilization solution was added to each eppendorf and then vortexed for20 minutes and then rinsed 7 times with sterile H20. Then the seeds weredried and then spread onto the surface of the MSO plates, covered andwrapped in parafilm. Put plates at 26° C. with a 16 hour photoperiod.For germination of T1, 1.2 ml of sterilization solution was added andsterilized as above, except the dry seeds were spread onto MSO platedwith 500 μg/ml spectinomycin (Petit Havana) and 350 μg/ml to select fortransformants (Kumar and Daniell, 2004).

Preparation of Tobacco Tissue Culture Media (RMOP and MSO Media).

The shoot-inducing RMOP media was made by adding one packet MS saltsmixture, 30 gm sucrose, 1 ml benzylaminopurine, BAP (1 mg/ml stock); 100ul napthalene acetic acid, NAA (1 mg/ml stock), 1 ml thiaminehydrochloride (1 mg/ml stock) to 1 L dH2O. The pH was adjusted to 5.8with 1N KOH and 7.0 g/L phytagar was added to the mixture which wasautoclaved, cooled and plated out under the hood (Kumar and Daniell,2004). The root-inducing MSO media was prepared by adding 30 g sucroseand one packet (4.3 g) of Murashige & Skoog (MSO) salt mixture (GibcoBRL) to 1 L dH2O. The pH was adjusted to 5.8 with 1N KOH, then 7 g/Lphytagar was added and the mixture was autoclaved (Kumar and Daniell,2004).

Biolistic Transformation of Tobacco Leaf Chloroplast.

About 4 weeks prior to the planned bombardment, wild-type(untransformed) tobacco plants were micropropagated from seeds usingsterile techniques. Two varieties of tobacco were generated for thebombardment: Petit Havana (model) and LAMD-609 (low nicotine hybridproduced by backcrossing a Maryland type variety, MD-609, to alow-nicotine producing burley variety, LA Burley 21 (Collins et al.,1974).

Preparation of the Gold Particles and DNA/Particle Suspension.

Fifty mg of gold particles (0.6 μm) were placed in a micro centrifugetube and 1 ml of freshly prepared 70% ethanol was added. The mixture wasvortexed for 3-5 minutes and incubated at room temperature for 15minutes. The gold particles were pelleted by spinning for 5 seconds andthen the supernatant was discarded. 1 ml of H2O was added to theparticles and vortexed for a minute. Particles were allowed to sit for 1minute and pulse centrifuged for 3 seconds. The supernatant wasdiscarded and this was repeated three times. After the last spin, 50%glycerol was added to a concentration of 60 mg/ml. The gold particleswere stored at −20° C. (Kumar and Daniell, 2004).

Coating DNA onto Macrocarriers.

The gold particles prepared in 50% glycerol (60 mg/ml) were vortexed for5 minutes to resuspended the particles. Fifty ul of gold particles wasremoved and placed in a micro centrifuge tube. 10 ul (1 μg/nl) of thepLD-AB-NS3 vector DNA was added and quickly vortexed. Then, 50 ul offreshly prepared 2.5M CaCl2 (367.5 mg of CaCl2 into 1 ml of d/aH2O) wasadded and vortexed. Finally, 0.1M spermidine-free base (20 ul) was addedand the tube was vortexed for 20 minutes at 4° C. 200 ul of absoluteethanol added to each tube and centrifuged for 2 seconds, then theethanol was discarded. The wash was repeated 4 times. After the washes,the particles were resuspended in 40 ul of absolute ethanol and kept onice (Kumar and Daniell, 2004).

Preparing the Biolistic Gun and Consumables.

Stopping screens, rupture disk holders, macrocarrier holders wereautoclaved to ensure that they were sterile. Rupture disks andmacrocarriers were washed in 50 ml of autoclaved H2O and 70% ethanol.The Bio-Rad PDS-1000/He (gene gun) shelves, macrocarrier holder, rupturedisk holder were washed with 70% ethanol. After the pump under the hoodwas turned on, the main valve on the helium tank was opened and thevalve controlling pressure to the gene gun was set to 13500 psi (Kumarand Daniell, 2004).

Bombardment.

The bombardment was performed as described previously (Daniell, 1997).Stopping screens were placed in macrocarrier holders. 6 ul of particlemixture was spread evenly onto the macrocarrier. The gold suspension wasallowed to dry. One rupture disk was placed in the holder ring andscrewed in place at the top of the vacuum chamber. The stopping screenand macrocarrier with the gold/DNA (in holder) were placed into theretaining assembly. The assembly was placed into the vacuum chamber. Apiece of sterile whatman #1 filter paper was placed on solidified RMOPmedia in a petri dish. One leaf at a time was placed on the whatmanpaper abaxial side upwards. The petri dish with leaf was placed on aplastic holder and placed in the next to last slot in the vacuumchamber. The chamber door was closed and secured. The power switch forthe gene gun was turned on. A vacuum was allowed to build to 28 psi inthe bombardment chamber. When 28 psi was reached, the fire switch waspressed until the rupture disk ruptured (u1100 psi). After delivery ofthe gold particles with vector DNA, the vacuum was released and thePetri dish taken out and covered. The petri dishes were wrapped inaluminum foil and kept in the dark for 48 hours at room temperature torecover from the shock of bombardment (Kumar and Daniell, 2004).

Selection and Regeneration of Transgenic Lines. After recovering in thedark for 48 hours from bombardment, leaves were cut into 5 mm2 squaresand placed on a petri dish containing RMOP media containingspectinomycin. For Petite Havana, 500 ug/ml of spectinomycin was usedand for LAMD-609, 350 ug/ml of spectinomycin was used for the firstround of selection (with the abaxial side down). Four to six weeks laterwhen the shoots appeared, they were cut into 2 mm2 pieces andtransferred to fresh RMOP media with spectinomycin for the second roundof selection (500 ug/ml for Petite Havana and 350 ug/ml for LAMD-609).During the second selection, the shoots that appeared and testedpositive for cassette integration into the chloroplast by PCR analysiswere grown in sterile glass jars containing fresh media withspectinomycin until the shoots grew to fill the jar. Then the plantswere transferred to pots with soil containing no antibiotic. Pottedplants were grown in a 16 hour light/8 hour dark photoperiod in thegrowth chamber at 26° C. (Kumar and Daniell, 2004).

Isolation of total plant genomic DNA from Tobacco Leaf.

The QIAGEN's DNeasy® Plant Mini Kit was used for isolating the total DNAfrom plant tissue as described in the Qiagen manual. 100 mg of thetissue was grounded in liquid nitrogen to a fine powder and wastransferred to a cooled eppendorf and 400 ul of Buffer AP1 and 4 ul ofRNase A stock solution (100 mg/ml) was added and vortexed. The mixturewas incubated for 10 minutes at 65° C. and mixed about 2-3 times duringincubation by inverting the tube. 130 ul of Buffer AP2 was added to thelysate, mixed, and then incubated on ice for 5 minutes. The lysate wasapplied to the QIAshredder spin column (lilac) sitting in a 2 mlcollection tube and then centrifuged for 2 minutes. The flow-through wastransferred to a new tube and 1.5 volumes of buffer AP3/E were added tothe lysate and mixed immediately. 650 ul of the mixture was applied tothe DNeasy mini spin column sitting in a 2 ml collection tube and thencentrifuged for 1 minute at 8000 rpm. The DNeasy column was placed in anew 2 ml collection tube and 500 ul Buffer AW was added to the DNeasycolumn and centrifuged for 1 minute at 8000 rpm. The flow-through wasdiscarded and collection tube was reused in the next step. 500 ul BufferAW was added to the DNeasy column and centrifuged for 2 minutes atmaximum speed to dry the membrane. The DNeasy column was transferred toa 2 ml microcentrifuge tube and 100 ul of preheated (65° C.) Buffer AEwas directly added onto the DNeasy membrane. The membrane was incubatedfor 5 minutes at room temperature and then centrifuged at 8,000 rpm for1 minute to elute the DNA. The DNA was kept at −20° C. for use in PCRand Southern analysis.

PCR Analysis of Integration into the Chloroplast Genome.

To confirm the transgene cassette integration into the chloroplastgenome, two primers sets were designed and assigned numbers with theplus (P) being for the forward primer and minus (M) being for thereverse primer. The 3P/3M (3P: 5′-AAAACCCGTCCTCCGTTCGGAT-TGC-3′) primerannealed to anneal to a unique portion of the chloroplast genome and 3M(5′-CCGCGTTGTTTCATCAAGCCTTACG-3′) annealed to the integrated aadA gene(Daniell et al, 2001b). For the PCR reaction, 200 ng of plant DNA, 5 μlof 10× buffer, 4 μl of 2.5 mM dNTP, 2 μl of each primer from the stock,0.50 Taq DNA polymerase and H2O to make up the total volume to 50 ul.The amplification was carried for 25 cycles of the following reaction:94° C. for 5 mins, 94° C. for 45 sec, and 65° C. for 45 sec, 68° C. for1.5 min, 68° C. for 7 mins. To confirm the integration of gene ofinterest, PCR was performed using primer pairs 5P(5′-CTGTAGAAGTCACCATTGTTGTGC-3′ and 2M (5′-TGACTGCCCACCTGAGAGCGGACA-3′).The amplification was carried during 25 cycles of the followingreaction: 95° C. for 5 mins, 95° C. for 1 min, and 68° C. for 1 min, 72°C. for 3 min, 72° C. for 10 mins. 5 ul of each PCR products includingthe controls were loaded into a 0.8% agarose gel to confirm the results.pLD-NS3 was used as the positive control and wild type petite Havana wasused as a negative control.

Southern Blot Analysis.

These steps were performed as described in (Daniell et al., 2004a). Thetotal DNA isolated from T0 plants as well as from untransformed tobaccoplants with QIAGEN's DNeasy® Plant Mini Kit was digested as follows: 10ul (2 ug) DNA from DNeasy, 3 μl of 10× buffer 3, 2 μl BglII enzyme(NEB), 14.7 μl sterile H2O, to a total volume of 30 μl. The digest wasincubated O/N at 37° C. The digestion was separated on a 0.8% agarose at50V for 3.5 hours. The gel was observed under UV light to verify thecomplete digestion of the plant DNA. The gel was soaked in 0.25N HCl(depurination solution) for 15 minutes in a continuous agitation. Thedepurination solution was discarded, and the gel was rinsed 2 times withsterile H2O for 5 minutes. The gel was then soaked in transfer buffer ona rotary shaker for 20 minutes. The transfer apparatus was assembled forthe transfer of the DNA to Duralon-UV nylon membrane. Four pieces of theWhatman paper were cut slightly larger than the gel and the membrane.Two pieces of Whatman paper were dipped into the transfer solution andplaced on three sponges placed in a large pyrex dish partially filledwith transfer buffer. The gel was removed from the transfer buffer andinverted on the Whatman paper. The nylon membrane was soaked in waterand then placed on the gel. Removed air bubble gently and arrangedparafilm along all the side to prevent horizontal DNA transfer. A stackof ordinary paper towels onto the top of Whatman filter paper and thenadded a 500 g weight to encourage transfer. From the bottom of the pyrexdish the transfer was in the following order: sponges, 2 filter paper,gel, parafilm at edges, nylon membrane, 2 filter paper, paper towels andweight. The set up was left for transfer over night and the next day themembrane was washed on 2×SSC (3M NaCl, 0.3M Na citrate, H2O, the pH wasadjusted with 1N HCl to 7 and water was added to 1 L) for 5 minutes. Themembrane was air-dried and then cross-linked using the GS Gene Linker UVChamber (BIO-RAD) at the C3 setting.

Generating and Labeling Probes.

The probes were prepared by the random primed ³²P-labeling (Ready-to-goDNA labeling beads, Amersham Pharmacia).A pUC universal vectorcontaining the chloroplast flanking sequences was used to generate theflanking probe. The restriction digest was set-up as follows: 20 μl ofpUC-ct, 1 μl 10× buffer 3, 1 μl BamHI (NEB), 1 μl BglII (NEB), 0.3 μl ofBSA, 6.7 μl of sterile H2O to a total volume of 30 μl. The reaction wasincubated overnight at 37° C. The restriction digest for the genespecific probe was as follows: 20 μl of pLD-AB-NS3, 1 μl of EcoRI (NEB),1 μl of EcoRV (NEB), 3 μl of 10× buffer #3 (NEB), 0.3 ul of BSA, 4.7 μlsterile H2O to a total volume of 30 The reaction was incubated 0/N at37° C. 45 μl of each probe was denatured at 94° C. for 5 minutes andthen placed on ice for 3 minutes. The probes were added to the ready mixtube (Quantum G-50 Micro columns, Amersham) and gently mixed byflicking. 5 μl of α32P was added to the ready mix tube and then it wasincubated at 37° C. for 1 hour. The resin in the G50 column wasresuspended by vortexing. The cap was loosened and the bottom plugbroken off. Then the column was placed in a microcentrifuge tube withthe top cut off and centrifuged for 1 minute at 3000 rpm. The collectiontube with the supernatant was discarded and the column was transferredto a new tube. The probes were added to the center of the resin andcentrifuged for 2 minutes at 3000 rpm and then the column was discarded.The amount of labeled DNA probe to be used was determined.

Prehybridization, Hybridization and Washing of the Membrane.

For prehybridization, the membrane was washed with sterile water. TheQuick Hyb solution was gently mixed by inverting and warmed. Themembrane was placed in a bottle with the top facing in towards thesolution and 5 ml of the pre-Hyb solution was added and incubated for 60mins at 68° C. 100 μl of salmon sperm (10 mg/ml) was added to thelabeled probes and the mixture was heated at 94° C. for 5 minutes. Theprobes were added to the pre-Hyb solution and the blot was incubated for1 hour at 68° C. After hybridization, the membrane was removed from thebottle and washed twice in 50 ml of 2×SSC and 0.1% SDS for 15 minutes atroom temperature. Then, the membrane was washed twice in 50 ml ofpre-heated 0.1×SSC and 0.1% SDS for 15 minutes at 60° C. The membranewas then placed on top of Whatman filter paper for 30 minutes to dry andthen wrapped in saran wrap. The membrane was exposed to film overnight,stored at −80° C. and then developed.

Plant Expression of NS3 and Immunoblot Analysis.

Petit Havana and LAMD-609 leaf sections were cut and 100 mg plant leaftissue was weighed and grounded with liquid nitrogen in cold mortar andpestles and transferred to a microcentrifuge tube. Fresh plantextraction buffer (PEB: 60 ul of 5M NaCl, 60 ul of 0.5M EDTA (pH 8), 600ul of 1M Tris-HCl (pH 8), 2 ul of Tween-20, 30 ul of 10% SDS, 3 ul of 14mM β-mercaptoethanol (BME), 1.2 ml of 1M sucrose, 1 ml sterile H2O and120 ul of 100 mM PMSF) was made and kept on ice. To make 100 mM of PMSF,17.4 mg of powdered PMSF (Sigma) was weighed out, put into 1 ml ofmethanol and vortexed, and stored at up to 1 month at −20° C. 200 ul ofPEB was added to each plant sample on ice and then samples were mixedfor 3 minutes with a micropestle. The samples were centrifuged at 13,000rpm for 10 mins to obtain the supernatant containing the solubleproteins. 20 μl of these extracts were mixed with 20 μl of sampleloading buffer containing BME. Samples were then boiled for 5 minutesand loaded into SDS-PAGE gel. The procedure for the rest was identicalto the protocol for E. coli-expressed NS3 and Immunoblot Analysis (seeabove sections).

Enzyme Linked Immuno Sorbant Assay (ELISA).

The levels of NS3 in transgenic LAMD-609 were calculated as a percentageof the total soluble protein of leaf extracts. The quantification of NS3in the plant crude extract was done using the enzyme linkedimmunosorbant assay (ELISA). 100 mg of transgenic leaf samples (young,mature, old) and the wild type leaf samples (young, mature, old) werecollected. The leaf samples were collected from plants exposed toregular lighting pattern (16 h light and 8 h dark), 3 day continuouslight, and 5 day continuous light. The leaf samples were finely groundedin liquid nitrogen and the leaf powder was transferred into an eppendorftube. To extract the protein, plant protein extraction buffer (15 mMNa2CO3, 35 mM NaHCO3, 3 mM NaN3, pH: 9.6, 0.1% Tween, 5 mM PMSF) wasused to resuspended the leaf powder. In order to check the proteinconcentration, the standards, test samples and antibody were diluted incoating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3; pH: 9.6). Thestandards ranging from 50 to 500 ng/ml (500 ng/ml, 400 ng/ml, 300 ng/ml,200 ng/ml, 100 ng/ml and 50 ng/ml) were made by diluting purified NS3 incoating buffer (stock: 1000 ng/ml). The standards and protein samples(100 μl) were coated to 96-well polyvinyl chloride microtiter plate(Cellstar) for 1 h at 37 C followed by 3 washes with PBST and 2 washeswith water. Blocking was done with 3% fat-free milk in PBS and 0.1%Tween and incubated for 1 h followed by washing. The primary anti-NS3antibody (Neomarkers) diluted (1:500) in PBST containing milk powder wasloaded into wells and incubated for 1 h followed by washing steps andthen again incubated with 100 μl of anti-mouse goat-HRP conjugatedantibody (American Qualex, 1: 5000) diluted in PBST containing milkpowder. The plate was then incubated for 1 h at 37° C. After theincubation, the plate was washed thrice with PBST and twice with water.The wells were then loaded with 100 μl of 3,3,5,5-tetramethyl benzidine(TMB from American Qualex) substrate and incubated for 10-15 min at roomtemperature. The reaction was terminated by adding 50 μl of 2N sulfuricacid per well and the plate was read with a plate reader (DynexTechnologies) at 450 nm (Modified form of protocol from Ausubel et al.,4^(th) edition).

Bradford Assay for Protein Quantification (Bio-Rad Manual).

The Bradford assay was used to determine the total protein from theplant extracts prepared as described above. This was used to determinethe percent of NS3 antigen in the total soluble protein extract (or %TSP). An aliquot of plant extract as prepared above was thawed on ice.Extraction buffer (15 mM Na2CO3, 35 mM NaHCO3, 0.2 g NaN3, 0.1% Tween20, and 5 mM PMSF adjusted to pH 9.6) was used to make Bovine SerumAlbumin (BSA) standards ranging from 0.05 to 0.5 μg/μl. Plant extractswere diluted 1:20 and 1:30 with extraction buffer. 10 μl of eachstandard and 10 μl of each plant dilution were added to the wells of a96 well microtiter plate (Costar) in duplicates. Bradford reagent(Biorad protein assay) was diluted 1:4 with distilled water as specifiedand 200 μl was added to each well. Absorbance was read. The comparisonof the absorbance to known amounts of BSA to that of the samples wasused to estimate the amount of total protein.

Results

Construction of pLD-5′UTR/NS3 Vector for Tobacco ChloroplastTransformation.

The NS3 gene (starting 134 bp) in pcDNA3.1D/V5-His-TOPO was PCRamplified and the restriction sites, SacI and SnaBI at the 5′ end andNod at the 3′ end of the 134 bp of the NS3 gene were created for furthersubcloning. A PCR product of 134 bp in size was obtained byamplification. The PCR product was then digested with Sad and Nod andwas ligated between the same sites in p-Bluescript II KS vector. Thetransformed colonies were selected as the pBluescript vector containsthe LacZ gene for a complementation and blue/white selection. Theligated plasmid pBS-NS3 was isolated using midi-prep and the PCR productwas sequenced. The sequence was compared with the original NS3 sequencesent by Dr. Lasarte. After confirming that the 5′ of the NS3 gene(beginning 134 bp) was successfully cloned into pBluescript, theremaining NS3 gene (1770 bp) was digested from the originalpcDNA3.1D/V5-His-TOPO vector with BstXI and EcoRV and ligated betweenthe same sites in pBluescript vector. Therefore, the entire NS3 gene(1.9 kb) was cloned into p-Bluescript vector. The entire NS3 gene wasdigested with SnaBI and HindIII and cloned downstream of psbA 5′UTR inpCR2.1. Finally, the psbA 5′UTR and the NS3 gene were digested withEcoRV and EcoRI (fragment size 2.1 kb) from pCR2.1 and ligated into thefinal universal vector, pLD-AB-Ct. The 5.9 kb expression vector wasdeveloped with unique features facilitating the genetic engineering ofplant chloroplasts (FIG. 16). The integration of cloned chloroplast DNAinto the plastid genome occurs exclusively through site-specifichomologous recombination and excludes the foreign vector DNA (Kavanaghet al., 1999). The pLD-AB-Ct uses trnA and trnI genes (chloroplasttransfer RNAs coding for alanine and isoleucine) from the invertedrepeat region of the tobacco chloroplast genome as flanking sequencesfor homologous recombination (Daniell, 1999). This chloroplastexpression vector is considered universal because it can be used totransform the chloroplast genomes of not just tobacco, but several otherplant species as well (Daniell, 1999). Therefore, this pLD-AB-Ct wassuccessfully used as the backbone for the 5′UTR/NS3 cassette (FIG. 15).

Selection and Regeneration of Transgenic Lines.

After recovering in the dark for 48 hours from bombardment, leaves werecut into 5 mm2 pieces and placed on RMOP (Daniell, 1993) platescontaining 500 μg/ml spectinomycin for Petite Havana and 350 μg/ml forLAMD-650, for the first round of selection as described in Daniell(1997). From 10 bombarded Petit Havana leaves, 15 green shoots appearedafter 4 weeks. From 10 bombarded LAMD leaves, 3 green shoots appearedwithin 7 weeks, so the shoots from the low-nicotine tobacco took longerto sprout and were less numerous. Untransformed cells appeared bleachedon the antibiotic because they did not contain the aadA gene (FIG. 18).For second selection the shoots were cut into 2 mm² pieces and thentransferred to fresh RMOP plates with 500 μg/ml and 350 μg/mlspectinomycin for Petite Havana and LAMD spectinomycin respectively(FIG. 19).

During the second round of selection, the shoots that appeared andtested positive for cassette integration into the chloroplast genome byPCR analysis were grown in sterile jars containing fresh plant mediawith spectinomycin until the shoots grew to fill the jars (FIG.20A).Then the plants were transferred to pots with soil containing noantibiotic (FIG. 20B). Potted plants were grown in a 16 hour light/8hour dark photoperiod in the growth chamber at 26° C.

PCR Analysis of Transgenic Lines.

Two primer sets were used to identify transgenic lines. The 3P/3M set,the 3P primer annealed to the chloroplast genome outside of the insertedcassette and the 3M primer annealed to the chimeric aadA gene (FIG.21A). When both of the primers annealed, a 1.65 kb PCR product wasobserved, however, there was no PCR product in the untransformed (−)Petit Havana and LAMD line (FIG. 21B). In addition, no PCR productshould be observed if the foreign gene cassette was integrated into thenuclear genome or if the plants were mutants lacking the aadA gene. Outof the 7 putative transgenic lines shown, all 7 were positive forinsertion of the foreign gene cassette (FIG. 21B).

For the 5P/2M set, the 5P primer annealed to the chimeric aadA gene andthe 2P primer annealed to trnA gene within the cassette (FIG. 22A). Whenboth of the primers annealed, a 3.7 kb PCR product was observed,however, there was no PCR product in the untransformed (−) petit Havanaor LAMD line (FIG. 22B). The correct size of PCR product (3.7 kb)indicated that the entire foreign gene cassette and not just the aadAgene had been integrated into the chloroplast genome (FIG. 22A).

Southern Blot Analysis of Transgenic Plants (T0).

Southern blots were performed to confirm integration of the NS3 genecassette utilizing two different DNA probes (FIG. 23 and FIG. 24). A0.81 kb DNA fragment containing chloroplast-flanking sequences was usedto probe a Southern blot to determine homoplasmy or heteroplasmy afterbombardment with pLD-AB-NS3 (T0). This determination was also used toestimate chloroplasts genome copy number. BglII digested DNA fromtransformed plants produced a 5.2 kb and 2.7 kb fragment when probedwith the 0.81 kb probe that hybridizes to the trnI and trnA flankingsequences (FIG. 23). Untransformed plant DNA from both tobacco varietiesproduced only a 4.47 kb fragment, indicating no integration of foreignDNA. Transgenic plant DNA (T0) produced only the 5.2 and 2.7 kb fragmentin all transgenic plants indicating homoplasmy (contained onlytransformed chloroplast genomes).

The second probe used was a 2.1 kb 5′UTR/NS3 sequence that hybridized toa 2.7 kb fragment in transformed plants and no fragment was evident inuntransformed plants (FIG. 24). All transgenic plants produced a 2.7 kbfragment corresponding to the NS3 sequence (FIG. 24).

Chloroplast-Synthesized NS3 and Immunoblot Analysis.

Petit Havana and LAMD were bombarded with pLD-AB-NS3. Western blotanalysis was performed on the leaf cell extracts. The total plantprotein was separated using 10% sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). The NS3 protein was detected by mousemonoclonal antibody against NS3. Western blots detected NS3 protein at69 kDa using chemiluminescense (FIG. 25A).

Quantification of Chloroplast-Synthesized NS3 by ELISA.

To quantify the amount of NS3 in transgenic Petit Havana and LAMD leafextracts, an indirect enzyme-linked immunosorbent assay (ELISA) wasused. The purified NS3 protein was used to make a six-point standardcurve. 1 μl of the plant protein extracts were diluted into 20 ul and 30ul of coating buffer to determine the dilution that would be in thelinear range of NS3 standard curve. The primary antibody was anti-NS3Mouse Monoclonal Antibody. The secondary antibody was Goat anti-mouseIgG conjugated to horseradish peroxidase. The addition of one stepsubstrate (TMB) into the wells resulted in a color change that waseventually read on a plate reader with a 450 nm filter. The totalsoluble protein (tsp) in the plant leaf extracts was determined with aBradford Bio-Rad Protein Assay. The levels of NS3 in transgenic LAMDwere calculated as a percentage of the total soluble protein of leafextracts (FIG. 26).

Discussion

HCV vaccine development began recently with the use of recombinant HCVproteins as the immunogenic material (Choo et al., 1994). The initialcandidate HCV vaccine developed in 1994, derived from the envelopeglycoproteins (gpE1/E2) of HCV, with muramyl dipeptide adjuvants,induced high levels of neutralizing antibodies in chimpanzees andprovided protection in a proportion of animals challenged with low dosesof the homologous strain (Choo et al., 1994; Houghton et al., 1997). Inthe chimpanzees that were infected, the risk of persistent infectionseemed to be reduced. Little new information about this candidatevaccine is available. Additional studies of a recombinant E1/E2 proteinand peptide vaccine produced in insect cells (Esumi et al., 1999) alsosuggested that induced antibodies could neutralize low-level challengewith homologous HCV in the chimpanzee. In one DNA vaccine studyutilizing chimpanzees, a plasmid encoding the E2 HCV protein was used asimmunogen and elicited antibodies and immune response but on challengewith homologous HCV, sterilizing immunity could not be achieved (Formset al, 2000). Other approaches to vaccine development have included theincorporation of HCV proteins into recombinant viruses (Siler et al.,2002; Brinster et al., 2002), the synthesis of HCV-like particles ininsect cells (Lechmann et al., 2001), expression of thehypervariable-1-region of E2 in tobacco plants (Nemchinov et al., 2000)and DNA-based immunization (Brinster et al., 2001; Forns et al., 2000).Plant synthesized recombinant TMV/HCV HVR1 epitope/CTB induced a strongimmune response when mice were immunized intranasally (Nemchinov et al.,2001). Plants infected with a recombinant tobacco mosaic virusengineered to express the hypervariable region 1 (HVR1) of HCV, theHVR1/CTB chimeric protein elicited both anti-CTB and anti-HVR1 serumwhich specifically bound to HCV virus-like particles. The HCV HVR1epitope was also cloned into alfalfa mosaic virus (ALMV) coat proteinand expressed in transgenic tobacco plants. The Plant-derivedHVR1/ALMV-CP reacted with HVR1 and ALMV-CP specific monoclonalantibodies and immune sera from individuals infected with HCV (Nemchinovet al., 2001). A replication-deficient recombinant adenovirus expressingHCV NS3 protein was constructed. Mice immunized with this recombinantadenovirus were protected against challenge with a recombinant vacciniavirus expressing HCV polyprotein (Arribillaga et al, 2002).

The NS3 gene was introduced into pLD-Ct, the universal chloroplastexpression vector, which was developed with unique features thatfacilitate chloroplast genetic engineering (FIG. 16). The 5′untranslated region (UTR) of the plastid psbA gene and its promoter wereused to increase translation efficiency. The 5′UTR is involved inmRNA-rRNA interactions (between the mRNA ribosome-binding site and 16S rRNA 3′ end) and interactions with translational-activating proteins thatfacilitate loading onto ribosomes (Maliga, 2002). The psbA gene encodesthe D1 protein of photosystem II and is rapidly turned over in thechloroplasts (Eibl et al., 1999). The psbA 5′UTR is about 200 bp andcontains a promoter. The 3′ regulatory region (3′UTR) is important formRNA stability and functions as an inefficient terminator oftranscription. A unique short inverted repeat (IR) which can potentiallyfold into a stem loop structure at the 3′UTR probably act as a RNAprocessing signal rather than termination signal, playing a role in bothRNA 3′ end formation and stabilization (Hager and Bock, 2000). ThepLD-Ct contains a chimeric aadA gene as a selectable marker, whichencodes aminoglycoside 3′-adenylyltransferase. This enzyme catalyzes thecovalent modification of aminoglycoside-type antibiotics and therebyinactivates them. The aadA protein catalyses the covalent transfer of anAMP residue from ATP to spectinomycin, thereby converting the antibioticinto an inactive from (adenyl-spectinomycin) that no longer inhibitsprotein biosynthesis on prokaryotic 70S ribosomes present in thechloroplast. The aadA gene is driven by a portion of the constitutivepromoter of the chloroplast 16S rRNA operon (Prrn). The pLD-AB vectorintegrates the 16S rRNA promoter, aadA gene, 5′UTR, NS3 gene and 3′UTRcassette into the Inverted Repeat (IR) regions of the chloroplast genomebetween the homologous flanking sequences, trnI and trnA genes. The trnIand trnA intergenic spacer regions are highly conserved among higherplants (Guda et al., 2000). The pLD-Ct vector was constructed with amultiple cloning site downstream of the aadA gene and upstream of theTpsbA portion and flanked by chloroplast transfer RNA genes forisoleucine and alanine (trnI and trnA respectively). The plasmid canreplicate autonomously because it contains a unique chloroplast originof replication (Daniell, 1990; Kumar et al. 2004a, b) and ColE1 originof replication that operates in E. coli (Glick and Pasternak, 1998). Thetranslational apparatus of chloroplasts very much resembles that ofprokaryotes, in that tRNAs, rRNAs, ribosomal proteins and the initiationand elongation factors exhibit strong similarity with their counterpartsin E. coli (Brixey et al., 1997). As a way of testing the integrity ofthe NS3 cassette and its potential for protein expression, E. coli wastransformed with pLD-AB-NS3. Western blot analysis performed on the E.coli cell lysates indicated the presence of NS3 protein at the expectedsize of 69 kDa (FIG. 17), while the untransformed E. coli cell lysatesshowed no protein. Since the protein synthetic machinery of chloroplastsis similar to that of E. coli (Brixey et al., 1997), the positiveexpression of NS3 suggested that it could be successfully expressedwithin transgenic chloroplasts. Two varieties of tobacco were bombardedwith gold particles coated with pLD-AB-NS3 (Daniell, 1993). Petit Havanais the model tobacco variety because it is amenable to geneticengineering. The second variety of tobacco bombarded with pLD-AB-NS3 wasLAMD-609. This tobacco hybrid contains 0.06% nicotine (Collin et al.,1974), which is at least 50-fold lower than the Petit Havana tobacco(3-4%). Tobacco is the easiest plant to genetically engineer and iswidely used to test suitability of plant-based systems for bioproductionof recombinant proteins. Tobacco is ideal for transformation because ofits ease for genetic manipulation and is an excellent biomass producerand a prolific seed producer (up to one million seeds produced perplant). Bombarded leaves were placed on RMOP medium containing noantibiotics and allowed to recover from bombardment in the dark for 48hours (Daniell, 1993; Daniell, 1997; Daniell et al. 2004a).

After the recovery period, bombarded leaf discs were placed on selectiveplant medium containing 500 μg/ml of spectinomycin. Green shoots thatemerged from the part of the leaf disc in contact with the medium wereconsidered putative transformants because growth indicated that the aadAgene had been integrated into the chloroplast genome and was expressingfunctional enzyme. Each shoot (transgenic event) was subjected to asecond round of selection (500 μg/ml of spectinomycin) in an effort toensure that only transformed genomes existed in the cells of thetransgenic lines (homoplasmy). A heteroplasmic condition is unstable andwill result in loss of the transgene when the cell divides withoutselective pressure (Hager and Bock, 2000). A PCR method of screeningputative transformants was utilized to distinguish chloroplasttransformants from mutants and nuclear transformants (Daniell et al.2004a). Only those transgenic lines with the appropriately sized PCRproducts were used in further characterizations. The Southern blotanalysis utilized the integrity of DNA complimentary hybridization toidentify specific sequences in the various plant genomes. Differentpositive transgenic lines (T0) were tested to confirm site-specificintegration and to determine homoplasmy or heteroplasmy (FIGS. 26 & 27).The 810 bp flanking sequence probe confirmed that the NS3 gene cassettehad been integrated into the chloroplast genome. An enzyme-linkedimmunosorbent assay (ELISA) utilizing 96-well microtiter plates, wasused to quantify the amount of NS3 in transgenic LAMD-609 leaf extracts.The highest percentage of NS3 was 2% of total soluble protein, observedin the old leaves. In conclusion, this study reports successfulexpression of the HCV NS3 antigen in transgenic chloroplasts and theplant derived recombinant HCV vaccine antigen can potentially reduceexpenses normally associated with the production and delivery ofconventional vaccines and is a safe and inexpensive source for theproduction of HCV vaccine antigen.

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Example 4 Transgenic Chloroplast Expression of Soluble Modified GreenFlourescent Protein and Interferon Alpha-5 Fusion Introduction

The World Health Organization estimates that approximately 170 millionpeople worldwide are infected with hepatitis C virus (HCV), with 3-4million new cases each year, and that more than one third of the world'spopulation is infected with hepatitis B virus (HBV). A large majority ofHCV-infected patients have severe liver cirrhosis and currently there isno vaccine available for this disease. In addition, the rising cost oftreatment for severe illnesses calls for the more economical productionof therapeutic proteins. Alpha interferons have therapeutic uses, suchas the inhibition of viral replication and cell proliferation,enhancement of the immune response, and most recently, the treatment ofpatients suffering from HCV. The Food and Drug Administration approved aspecific subtype of interferon-α (IFNα2b) for the treatment of HCV. Inan effort to produce another subtype of interferon-α (IFNα5, kindlyprovided by Dr. Jesus Prieto, Universidad De Navarra, Pamplona, Spain)in large quantities and free of contaminants for possible treatmentoptions and oral delivery of HCV, a fusion of smGFP-IFNα5 has beenexpressed in transgenic chloroplasts of Nicotiana tabacum var. darkfire, by inserting the smGFP (745 bp) and IFNα5 (515 bp) genes into thechloroplast genome by homologous recombination. The pLD-BB1 vectorcontains smGFP with a C-terminal fusion to IFNα5 containing a furincleavage site between the fusion proteins. The genes were cloned into auniversal chloroplast vector, pLD-ctv containing the 16S rRNA promoter,aadA gene coding for the spectinomycin selectable marker, psbA 5′ & 3′untranslated regions to enhance translation in the light and trnI & trnAhomologous flanking sequences for site specific integration into thechloroplast genome. Chloroplast integration of the smGFP-IFNα5 genes wasconfirmed by PCR and Southern blot analysis. The smGFP-IFNα5 fusionprotein expression was confirmed by immunoblot analysis and smGFPexpression under UV light. Expression was quantified by ELISA. ThesmGFP-IFNα5 fusion protein is analyzed via in vivo studies. Theexpression of smGFP-IFNα5 transgenic chloroplasts will facilitate thedevelopment of a new and alternate treatment for HCV and possible oraldelivery options with a lower cost of production.

Materials and Methods

Construction of the pLD-BB1 Vector

The IFNα5 gene was kindly provided by Dr. Jesus Prieto, Universidad DeNavarra, Pamplona, Spain, within Escherichia coli expression vectordesignated pET-28b (Novagen). The smGFP gene was obtained from OhioState University, within the plasmid vector psmGFP. The vector wastransformed into Ultra competent XL1 Blue MRF′ Tetracycline (tet) E.coli cells (Stratagene) that were endonuclease negative. The recombinantDNA techniques were carried out as detailed in Sambrook et al., 1989.

Preparation of Competent Cells

Ultra competent XL1 Blue MRF′ (tet) E. coli cells were made competent byinoculating 50 ml of Luria Bertani (LB) broth (10 gr Tryptone, 5 gryeast extract, 5 gr NaCl, pH 7.0, dH₂0 to a liter) with 500 μl of cellsand incubating at 37° C. overnight while shaking at 225 rpm using theOrbit Environ Shaker (Lab-Line). Once the Optical Density (OD) readingat 600 nm was between 0.4 and 0.6, the cells were transferred to several14 ml falcon tubes, chilled on ice for 15 minutes. The cells werecentrifuged at 8500 rpm for 6 minutes at 4° C. The subsequent E. colipellet was resuspended in 25 ml of cold 50 mM CaCl₂, mixed by vortexingand then incubated on ice for 15 minutes. The cells were recentrifugedat 8500 rpm for 6 minutes at 4° C. The cells were resuspended into 1 mlof 50 mM CaCl₂ and 1 ml of 30% glycerol, then gently inverted 3 times.Competent cells were gently aliquoted into micro centrifuge tubes (200μl/tube) being sure to keep everything cold at all times. Competentcells were labeled and stored at −80° C.

Midi-Prep of psmGFP

Inoculated E. coli containing psmGFP into 50 ml of liquid LB broth in a250 ml flask. 25 μl of ampicillin (amp) stock (100 mg/ml) was added tothe 50 ml LB above so that only the amp-resistant plasmids would grow.The flask was covered with aluminum foil and put in shaker at 37° C. for16 hours to grow-up cells. 40 ml of the overnight culture wastransferred to a clean 50 ml screw-cap centrifuge tube and spun down.The cells were centrifuged for 5 minutes at 5000 rpm. The Bio-RadMidi-prep kit cat. #732-6120 was used for DNA isolation. The supernatantcontaining LB and cellular waste was discarded. 5 ml of cellresuspension solution was added to the pellet and vortexed until thecells were resuspended. 5 ml of cell lysis solution was added and mixedby inverting the tube 8 times. The solution turned from milky, lightbeige to clear, light beige. 5 ml of neutralization solution was addedto the clear, beige solution and then the solution became a whiteprecipitant. The solution was centrifuged for 10 minutes at 8000 rpm.The supernatant was poured into a new 50 ml screw-cap centrifuge tube.The quantum prep mix was resuspended by vigorously shaking. 1 ml of thequantum prep mix was added to the clear supernatant. The solution wasswirled for 30 seconds to mix and then centrifuged for 2 minutes at 8000rpm to pellet the plasmids. The supernatant containing contaminants wasdissolved by the quantum prep mix and the pelleted plasmids remained 10ml of wash buffer was added to pelleted plasmids and the matrix wasresuspended in the wash buffer by shaking. The solution was centrifugedfor 2 minutes at 8000 rpm and discarded the supernatant. The pellet wasthen resuspended in 600 μl of wash buffer and transferred to columns incollection tube provided by the kit. Columns were centrifuged for 30seconds at 12,000 rpm at 4° C. and flow-through was discarded. Columnwas centrifuged for an additional 2 minutes at 12,000 rpm and thencolumns were transferred to sterile microcentrifuge tubes. 300 μl ofTris-EDTA (TE: 1M Tris, pH 8.0, 0.5M EDTA) was added to the column andcentrifuged at 8000 rpm for 2 minutes at 4° C. The column wastransferred to a fresh microcentrifuge tube and the same above step wasrepeated. The DNA was stored at −20° C.

Mini-Prep of pET28-IFNα5 by Rapid Plasmid Isolation

After cells had been growing for 12-16 hours at 37° C. in LB brothcontaining antibiotic, 1.5 ml of the cell suspension was put into aneppendorf and centrifuged at 13,000 rpm for 5 minutes. The supernatantwas discarded. An additional 1.0 ml of the same cell suspension wasadded and the centrifugation was repeated and the supernatant wasdiscarded. The pellet was resuspended in 100 μl of Solution I (GTE: 50mM D-(+)-Glucose, 10 mM EDTA, 25 mM Tris, pH 8) and vortexed. 1 μl of100 mg/ml Rnase was added to each tube and pulse vortexed. 200 μl ofsolution II (0.2NaOH, 10% SDS) was added and mixed by gently inverting 6times. The mixture was left to sit for 3 minutes and then centrifuged at13,000 rpm for 10 minutes at 4° C. The solution was pipetted into afresh, labeled eppendorf. Then, added 1000 μl of cold 95% ethanol toeach supernatant and vortexed briefly. The supernatant was centrifugedat 13,000 rpm at 4° C. for 17 minutes. The supernatant was removed anddiscarded, being careful not to dislodge the beige plasmid DNA in bottomof eppendorf. 500 μl of 70% cold ethanol was added and centrifuged for 5minutes. The ethanol was removed and discarded and subsequently dried inthe speed. The plasmid concentration and quality of DNA was measured byspectrophotometer. The DNA was stored at −20° C.

IFNα5 Amplification by Polymerase Chain Reaction (PCR)

Two primers were designed to amplify IFNα5 and include a furin cleavagesite at the 5′ end of the IFNα5 gene with the forward primer containingan EcoRV site and the reverse primer containing a Not I site for furthersubcloning. Primers were ordered from LIFE TECHNOLOGIES. When theprimers arrived, they were reconstituted in TE to yield a 100 μM stockthat was stored at −20° C. The PCR reaction contained 1.0 μl ofpET28-IFNα5, 5 μl of 10×PCR buffer, 5.0 μl of 10 mM dNTP's, 0.5 μl offorward primer (Furin-IFNα5-F-EcoRV), 0.5 μl of reverse primer(Furin-IFNα5-R-NotI), 0.5 μl of Pfu polymerase and 36.5.0 μl ofRnase/Dnase Free H₂0 to a total volume of 50 The PCR was performed assuggested by the manufacturer using the Gene Amp PCR system 2400(Perkin-Elmer). Samples were carried through 30 cycles using thefollowing temperatures and times: 94° C. for 1 minute, 55° C. for 1minute, 72° C. for 1 minute. Cycles were preceded by denaturation at 94°C. for 3 minutes and followed by a 5 minute extension time at 72° C. Thefinal PCR products were separated on a 0.8% agarose gel at 60 voltsuntil dye reached bottom (about 50 minutes).

Extraction of the EcoRV/Furin/IFN α5/NotI PCR Product from the Gel

The QIAGEN QIA quick gel extraction kit was used to extract the PCRproducts from the agarose gel. The gel was placed on a flat UV lightsource and the appropriate DNA fragment (515 bp) was cut out using asterile razor blade. The excised fragment was placed into a previouslyweighed microcentrifuge tube and reweighed to determine by differenceweight of the cut out fragment. To the eppendorf containing thefragment, 3 volumes of Buffer QC to 1 volume of gel slice was added. Theeppendorf was incubated at 50° C. for 10 minutes until the agarosemelted and solublized. One volume of isopropanol was then added to theeppendorf. The mixture was added to a QIA quick spin column placed in acollection tube and then centrifuged for 1 minute at 12,000 rpm at roomtemperature. The flow-through was discarded and the column wascentrifuged for an additional minute as above. The flow-through wasdiscarded again and 750 μl of buffer PE was added to the column andcentrifuged as above. The column was placed in a sterile microcentrifugetube and 50 μl of elution buffer (EB) was added to the column andcentrifuged for 1 minute at 12,000 rpm to elute the DNA (PCR product).

Ligation of the EcoRV/Furin/IFN α5/NotI PCR Product into pBKS vector

PCR products were eluted from the gel. The PCR products and the pBKSvector were restriction digested with EcoRV and NotI For digestion ofpBKS, 2 μl of midi-prepped pBKS, 0.2 μl of 100× bovine serum albumin(BSA), 2 μl of 10× New England Bio-Labs (NEB) #3 buffer, 0.5 μl of EcoRV(NEB), 0.5 μl of Not I (NEB) and 14.8 μl of Rnase/Dnase Free H₂0 to atotal volume of 20 μl. This was done in duplicate to ensure enough DNAwas available for the ligation reaction. The reaction was incubated at37° C. overnight (0/N). For the PCR product digestion, 4 μl of purifiedPCR product (IFNα5), 0.2 μl of 100× bovine serum albumin (BSA), 2 μl of10× New England Bio-Labs (NEB) #3 buffer, 0.5 μl of EcoRV (NEB), 0.5 μlof Not I (NEB) and 10.8 μl of Rnase/Dnase Free H₂O to a total volume of20 This was done in duplicate to ensure enough DNA was available for theligation reaction. The reaction was incubated at 37° C. overnight (0/N).Pulse vortexed the pBKS digestion, then added 4 μl of 6× bromophenolblue (bpb: 0.25% bromophenol blue, 40% w/v sucrose in d/a H₂O to a totalvolume of 10 ml) to the digestion. Loaded all 24 μl into well of 0.8%electrophoresis-grade agarose gel diluted into 1×TAE running buffer andthen ran at 60 volts (V) for 60 minutes. Pulse vortexed the PCR productdigestion, then added 4 μl of 6×bpb. Loaded all 24 μl into the well of a0.8% agarose gel and electrophoresed at 60V for 60 minutes. Thelinearized pBKS DNA fragment and the PCR products were gel eluted. Theduplicates were combined and the volume was reduced by vacuum to 25 μl.Ligated EcoRV/Furin/IFNα5/NotI into pBKS to complete pBKS-IFNα5 vector.For the ligation reaction, 4 μl of the pBKS backbone, 10 μl PCR product,4 μl 5× Ligase Buffer (Invitrogen), 0.2 μl T4 Ligase (Invitrogen), 2 μlof Rnase/Dnase Free H₂O to a total reaction volume of 20 μl. Theligation mixture was incubated at 4° C., O/N. Transformed ligation mixcontaining pBKS-IFNα5 into competent XL1 Blue MRF′ (tet) E. coli cells.

Transformation of pBKS-IFNα5 into Competent XL1 Blue MRF′ (tet) E. coliCells

Took out 100 μl of competent cells from −80° C. freezer and thawed in anice bucket. 10 μl of DNA from ligation reaction was added to thecompetent cells and mixed gently. The mixture was allowed to stand onice for a total of 30 minutes, gently rocking tube back and forth every10 minutes. The cells were heat shocked at 42° C. for 45-50 seconds. Thecells were left on ice for 2 minutes. 900 μl LB broth was added to eachand incubated at 37° C. on 225 rpm shaker for 45 minutes. The cells werepelleted by centrifugation at 13,000 rpm for 45 seconds and 800 μl ofthe supernatant was discarded. The cells were resuspened in theremaining 100 μl of LB broth and plated out transformed anduntransformed (control) onto X-gal/IPTG LB/amp agar plates (1 liter LBbroth, 15 gr agar, 100 μg/ml ampicillin, pH 7) under the hood. Plateswere covered and incubated 0/N at 37° C.

Selecting for Transformants

The pBKS cloning vector has a ColE1 origin of replication, ampicillinresistance, a DNA segment containing the lac promoter and theβ-galactosidase α-fragment (lacZ). Within this coding region is amultiple cloning site that does not disrupt the reading frame, but mustbe used in a host cell that codes for the carboxy-terminal portion ofthe β-galactosidase gene so that an enzymatically active β-galactosidaseprotein can be formed. The cells that grow due to this α-complementioncan be visually selected through a chromogenic test. Insertion of afragment of foreign DNA into the multicloning site of pBKS almostinvariably results in production of an amino-terminal fragment that isnot capable of α-complemention. Selective plates were made with LB agarand 100 μg/ml of ampicillin About 1 hour before transformation wascomplete, 40 μg/ml of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal)was spread onto the top of the plates while under the hood. X-gal is alactose analog that turns dark blue when it is hydrolyzed byβ-galactosidase. After the X-gal dried (about 15 minutes), 40 μl of 100mM of isopropyl-β-D-thiogalactoside (IPTG) was spread onto the plates.IPTG, another lactose analog, is a strong inducer of lacZ transcriptionbut is not digested/hydrolized by β-galactosidase. The plates werewarmed 37° C. for 30 minutes and then the plates were streaked with 100μl of the transformed bacterial cells were spread over the top of theagar. Allowed the plates to dry for 5 minutes, then incubated the platesin an inverted position at 37° C. overnight. Stored the plates afterincubation at 4° C. for 3 hours to allow the blue color from thechromogenic process to develop fully. Colonies without an interruptinginsert were blue because they had an active β-galactosidase. Colonieswhich had incorporated the insert were all white. These were picked toculture at 37° C. overnight and miniprepped as described in the previoussection. The DNA was stored at −20° C. The vector was confirmed byrestriction digestion analysis.

Amplification of smGFP by Polymerase Chain Reaction (PCR)

Two primers were designed to amplify smGFP and include specificrestriction sites with the forward primer containing a HindIII and SnaBIsite and the reverse primer containing an EcoRV site for furthersubcloning. Primers were ordered from LIFE TECHNOLOGIES. When theprimers arrived, they were reconstituted in TE to yield a 100 μM stockthat was stored at −20° C. The PCR reaction contained 3.0 μl of psmGFP,5 μl of 10×PCR buffer, 1.0 μl of MgSO₄, 5.0 μl of 10 mM dNTP's, 1.0 μlof forward primer (smGFP-F-HindIII-SnaBI), 1.0 μl of reverse primer(smGFP-R-EcoRV), 0.5 μl of Pfx polymerase and 33.5.0 μl of Rnase/DnaseFree H₂O to a total volume of 50 μl. The PCR was performed as suggestedby the manufacturer using the Gene Amp PCR system 2400 (Perkin-Elmer).Samples were carried through 30 cycles using the following temperaturesand times: 94° C. for 15 seconds, 50° C. for 30 seconds, 68° C. for 1minute. Cycles were preceded by denaturation at 94° C. for 5 minutes andfollowed by a 7 minute extension time at 68° C. After PCR, the vialswere placed on ice and 1 unit of Taq polymerase was added to each tubeand mixed. The vials were incubated at 72° C. for 10 minutes. The finalPCR products were separated on a 0.8% agarose gel at 60 volts for about50 minutes. The PCR product was then PCR purified using the QIAquick PCRpurification kit (Qiagen).

Ligation of the smGFP PCR Product into pCR®2.1-TOPO®

Thermus aquaticus (Taq) polymerase has non-template dependent activitywhich preferentially adds a single deoxyadenosine (A) to the 3′-ends ofa double stranded DNA molecule; therefore, most of the molecules PCRamplified possess single 3′ A overhang. The linearized vector suppliedwith the kit has a single, overhanging 3′ deoxythymidine (T) whichallows the PCR product to ligate efficiently with the vector. TA cloningutilizes the complementarity between the PCR product 3′-A overhangs andvector 3′-T overhangs and is one of the simpliest and most efficientmethods for the cloning of PCR products (Zhou, 2000). The PCR productswere ligated into pCR®2.1-TOPO® cloning vector (Invitrogen, 2000) thatcontained multiple restriction sites facilitating further subcloning. 2μl of psmGFP PCR product was combined with 1 μl of dilute salt solution,2 μl of Rnase/Dnase Free dH₂0 and 1 μl of the pCR®2.1-TOPO® cloningvector. The solution was gently mixed and incubated 5 minutes at roomtemperature. Chemically competent E. coli cells (TOP10) were taken from−80° C. freezer and thawed on ice. The transformation was startedimmediately after cells thawed. The cells were removed and gentlypipetted into a cold eppendorf on ice. 2 μl of the ligation mixture wasadded to an eppendorf containing the chemically competent cells andmixed gently without pipetting up and down. Then, the mixture wasincubated on ice for 30 minutes. Heat shocked the cells for 30 secondsat 42° C. without any shaking. The mixture was immediately transferredto ice. Then 250 μl of warm SOC broth was added and allowed to incubatein the shaker horizontally (200 rpm) for 1 hour at 37° C.

Selecting for Transformants

The pCR®2.1-TOPO® cloning vector has a ColE1 origin of replication,kanamycin resistance, ampicillin resistance and a DNA segment containingthe first 146 amino acids of the β-galactosidase gene (lacZ). Selectiveplates were made with LB agar and 50 μg/ml of kanamycin. About 1 hourbefore transformation was complete, 40 μg/ml of5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) was spread onto thetop of the plates while under the hood. X-gal is a lactose analog thatturns dark blue when it is hydrolyzed by β-galactosidase. After theX-gal dried (about 15 minutes), 40 μl of 100 mM ofisopropyl-β-D-thiogalactoside (IPTG) was spread onto the plates. IPTG,another lactose analog, is a strong inducer of lacZ transcription but isnot digested/hydrolized by β-galactosidase. The plates were warmed 37°C. for 30 minutes and then the plates were streaked with 150 μl of thetransformed bacterial cells were spread over the top of the agar.Allowed the plates to dry for 5 minutes, then incubated the plates in aninverted position at 37° C. overnight. Stored the plates afterincubation at 4° C. for 3 hours to allow the blue color from thechromogenic process to develop fully. Colonies without an interruptinginsert were blue because they had an active β-galactosidase. Colonieswhich contained an insert were all white so these were picked toculture. The culture was grown overnight at 37 C and miniprepped asdescribed in the previous section. The DNA was confirmed by restrictiondigestion analysis and stored at −20° C.

Building the pCR®2.1-5′UTR Vector

Two primers were designed to amplify 5′UTR and include specificrestriction sites with the forward primer containing an EcoRI site andthe reverse primer containing an EcoRV site for further subcloning.Primers were ordered from LIFE TECHNOLOGIES. When the primers arrived,they were reconstituted in TE to yield a 100 μM stock that was stored at−20° C. The PCR reaction contained 1.0 μl of template DNA, 5 μl of10×PCR buffer, 1.0 μl of MgSO₄, 5.0 μl of 10 mM dNTP's, 1.0 μl offorward primer (5′UTR-F-EcoRI), 1.0 μl of reverse primer(5′UTR-R-EcoRV), 0.5 μl of Pfx polymerase and 33.5.0 μl of Rnase/DnaseFree H₂0 to a total volume of 50 μl. The PCR was performed as suggestedby the manufacturer using the Gene Amp PCR system 2400 (Perkin-Elmer).Samples were carried through 30 cycles using the following temperaturesand times: 94° C. for 15 seconds, 50° C. for 30 seconds, 68° C. for 30seconds. Cycles were preceded by denaturation at 94° C. for 5 minutesand followed by a 7 minute extension time at 68° C. After PCR, the vialswere placed on ice and 1 unit of Taq polymerase was added to each tubeand mixed. The vials were incubated at 72° C. for 10 minutes. The finalPCR products were separated on a 0.8% agarose gel at 60 volts for about50 minutes. The PCR product was then PCR purified using the QIAquick PCRpurification kit (Qiagen). The purified PCR product was cloned intopCR®2.1-TOPO® cloning vector (Invitrogen, 2000) as described in previoussection. The transformants were selected and mini-prepped as describedin previous section. The vector was confirmed by restriction digestionanalysis.

Building the pLD-5′UTR Vector

A scraping of an E. coli glycerol stock containing the pLD expressionvector which was developed by Lee and Daniell was grown up in 50 ml ofliquid LB broth in a 250 ml flask. 25 μl of ampicillin (amp) stock (100mg/ml) was added to the 50 ml LB broth. The flask was covered and put inshaker at 37° C. for 16 hours to grow-up cells. A midi-prep wasperformed as described in previous section. The vector was confirmed byrestriction digestion analysis. For further subcloning, a restrictiondigestion was set up: 5.0 μl of pCR®2.1-5′UTR vector, 0.2 μl of 100×bovine serum albumin (BSA), 2.0 μl of 10× New England Bio-Labs (NEB) #3buffer, 0.5 μl of EcoRV (NEB), 0.5 μl of EcoRI (NEB) and 11.8 μl ofRnase/Dnase Free H₂O to a total volume of 20 This was done in duplicateto ensure enough DNA was available for the ligation reaction. Thereaction was incubated at 37° C. overnight (0/N). For the pLDrestriction digestion, 1.0 μl of pLD vector, 0.2 μl of 100× bovine serumalbumin (BSA), 2 μl of 10× New England Bio-Labs (NEB) #3 buffer, 0.5 μlof EcoRV (NEB), 0.5 μl of EcoRI (NEB) and 14.8 μl of Rnase/Dnase FreeH₂0 to a total volume of 20 μl. This was done in duplicate to ensureenough DNA was available for the ligation reaction. The reaction wasincubated at 37° C. for 1 hour. Pulse vortexed the digestions, thenadded 4 μl of 6× bromophenol blue (bpb: 0.25% bromophenol blue, 40% w/vsucrose in d/a H₂O to a total volume of 10 ml) to each digestion. Loadedall 24 μl of each digestion into separate wells of 0.8%electrophoresis-grade agarose gel diluted into 1×TAE running buffer andthen ran at 80 volts (V) for 60 minutes. The linearized pLD vector andthe 5′-UTR DNA fragments were gel eluted in 50 μl of Rnase/Dnase FreeH₂0 and vacuum evaporated to a volume of 15 μl. Ligated the 5′UTR DNAfragment into pLD vector to complete pLD-5′UTR vector. For the ligationreaction, 15 μl of the pLD backbone and 5′UTR DNA fragment combined, 4μl 5× Ligase Buffer (Invitrogen), and 1.0 μl T4 Ligase (Invitrogen) to atotal reaction volume of 20 μl. The ligation mixture was incubated at14° C., O/N. Transformed ligation mix containing pLD-5′UTR intocompetent XL1 Blue MRF′ (tet) E. coli cells using SOC broth instead ofLB broth as described in previous section. The transformation reactionwas plated out onto LB/amp agar plates (1 liter LB broth, 15 gr agar,100 μg/ml ampicillin, pH 7) under the hood. Plates were covered andincubated 0/N at 37° C. 10 bacterial colonies were selected and cultured0/N at 37° C. and mini-prepped as described in the previous section. TheDNA was confirmed by restriction digestion analysis and stored at −20°C.

Building the pBKS-smGFP-IFNα5 Vector

The pBKS-IFNα5 vector and the pCR®2.1-smGFP vector were thawed on ice.For further subcloning, a restriction digestion was set up: 2.0 μl ofpCR®2.1-smGFP vector, 1.0 μl of 1× bovine serum albumin (BSA), 2.0 μl of10× New England Bio-Labs (NEB) #2 buffer, 1.0 μl of EcoRV (NEB), 1.0 μlof HindIII (NEB) and 12.0 μl of Rnase/Dnase Free H₂0 to a total volumeof 20 μl. This was done in duplicate to ensure enough DNA was availablefor the ligation reaction. The reaction was incubated at 37° C. for 2hours. For the pBKS-IFNα5 vector restriction digestion, 2.0 μl ofpBKS-IFNα5 vector, 1.0 μl of 1× bovine serum albumin (BSA), 2 μl of 10×New England Bio-Labs (NEB) #2 buffer, 1.0 μl of EcoRV (NEB), 1.0 μl ofHindIII (NEB) and 12.0 μl of Rnase/Dnase Free H₂0 to a total volume of20 μl. This was done in duplicate to ensure enough DNA was available forthe ligation reaction. The reaction was incubated at 37° C. for 2 hours.Pulse vortexed the digestions, then added 4 μl of 6× bromophenol blue(bpb: 0.25% bromophenol blue, 40% w/v sucrose in d/a H₂O to a totalvolume of 10 ml) to each digestion. Loaded all 24 μl of each digestioninto separate wells of 0.8% electrophoresis-grade agarose gel dilutedinto 1×TAE running buffer and then ran at 80 volts (V) for 60 minutes.The linearized vector and the smGFP DNA fragments were gel eluted in 50μl of Rnase/Dnase Free H₂0 and vacuum evaporated to a volume of 15 μl.Ligated the smGFP DNA fragment into pBKS-IFNα5 vector to completepBKS-smGFP-IFNα5 vector. For the ligation reaction, 15 μl of thepBKS-IFNα5 backbone and smGFP DNA fragment combined, 4 μl 5× LigaseBuffer (Invitrogen), and 1.0 μl T4 Ligase (Invitrogen) to a totalreaction volume of 20 μl. The ligation mixture was incubated at 14° C.,O/N. Transformed ligation mix containing pBKS-smGFP-IFNα5 vector intocompetent XL1 Blue MRF′ (tet) E. coli cells using SOC broth instead ofLB broth as described in previous section. The transformation reactionwas plated out onto LB/amp agar plates (1 liter LB broth, 15 gr agar,100 μg/ml ampicillin, pH 7) under the hood. Plates were covered andincubated 0/N at 37° C. 10 bacterial colonies were selected and cultured0/N at 37° C. Then, a mini-prep was performed as described in theprevious section. The DNA was confirmed by restriction digestionanalysis and stored at −20° C.

Building the pLD-5′UTR-smGFP-IFNα5 Vector

The pBKS-smGFP-IFNα5 vector and the pLD-5′UTR vector were thawed on ice.For further subcloning, a restriction digestion was set up: 5.0 μl ofpLD-5′UTR vector, 2.0 μl of 10× New England Bio-Labs (NEB) #3 buffer,2.0 μl of 1× bovine serum albumin (BSA) 1.0 μl of NotI (NEB), 1.0 μl ofEcoRV (NEB) and 9.0 μl of Rnase/Dnase Free H₂O to a total volume of 20μl. This was done in duplicate to ensure enough DNA was available forthe ligation reaction. The reaction was incubated at 37° C. for 1 hour.For the pBKS-smGFP-IFNα5 restriction digestion, 5.0 μl ofpBKS-smGFP-IFNα5 vector, 2.0 μl of 1× bovine serum albumin (BSA), 2 μlof 10× New England Bio-Labs (NEB) #4 buffer, 1.0 μl of NotI (NEB), and10.0 μl of Rnase/Dnase Free H₂O to a total volume of 20 μl. This wasdone in duplicate to ensure enough DNA was available for the ligationreaction. The reaction was incubated at 37° C. for 1 hour. Then, 1.0 μlof SnaBI (NEB) was added and the reaction was incubated an additional 1hour at 37° C. Pulse vortexed the digestions, then added 4 μl of 6×bromophenol blue (bpb: 0.25% bromophenol blue, 40% w/v sucrose in d/aH₂O to a total volume of 10 ml) to each digestion. Loaded all 24 μl ofeach digestion into separate wells of 0.8% electrophoresis-grade agarosegel diluted into 1×TAE running buffer and then ran at 80 volts (V) for60 minutes. The linearized pLD-5′UTR vector and the smGFP-IFNα5 DNAfragments were gel eluted in 50 μl of Rnase/Dnase Free H₂O and vacuumevaporated to a volume of 15 μl. Ligated the smGFP-IFNα5 DNA fragmentinto pLD-5′UTR vector to complete pLD-BB1 vector(pLD-5′UTR-smGFP-IFNα5). For the ligation reaction, 15 μl of thepLD-5′UTR backbone and smGFP-IFNα5 DNA fragment combined, 4 μl 5× LigaseBuffer (Invitrogen), and 1.0 μl T4 Ligase (Invitrogen) to a totalreaction volume of 20 μl. The ligation mixture was incubated at 14° C.for four hours. Transformed ligation mix containing pLD-BB1 intocompetent XL1 Blue MRF′ (tet) E. coli cells using SOC broth instead ofLB broth as described in previous section. The transformation reactionwas plated out onto LB/amp agar plates (1 liter LB broth, 15 gr agar,100 μg/ml ampicillin, pH 7) under the hood. Plates were covered andincubated 0/N at 37° C. 15 bacterial colonies were selected and cultured0/N at 37° C. and mini-prepped as described in the previous section. Theremaining 500 μl of each bacterial 0/N cultures were placed on ice. TheDNA was confirmed by restriction digestion analysis and stored at −20°C. Four positive clones were selected and the corresponding bacterialculture on ice was use to inoculate 50 ml of liquid LB broth/Amp/Spec(100 μg/ml of Ampicillin; 100 mg/ml Spectinomycin) in a 250 ml flask andcovered. The cultures were placed in a shaker and incubated at 37° C.for 16 hours. The cultures were midi-prepped as described previously.The DNA was confirmed by restriction digestion analysis and stored at−20° C. Glycerol stocks were also made and stored at −80° C.

E. coli Expression of smGFP-IFNα5 and Immunoblot AnalysisExtraction of Protein from Transformed E. coli Cells

E. coli containing pLD-smGFP-IFNα5 was scraped off the top of theglycerol stock under the hood and inoculated 5 ml of Terrific Broth (TB)containing 25 μl of 100 mg/ml spectinomycin. Untransformed E. coli cellswere added to 5 ml of Terrific Broth (TB) as a negative control. Theinoculated broths were incubated in a shaker at 37° C. for 16 hours. 800μl of cultured cells were placed in an eppendorf tube and centrifugedfor 2 minutes. The supernatant was discarded. The pelleted cells werewashed with 1 ml of 1× Phosphate-Buffered Saline (PBS: 140 mM NaCl,2.7Mm KCl, 4 mM Na₂HPO₄, 1.8 mM KH2PO4, pH 7.2) resuspend the pellet.Then, the suspension was centrifuged for 1 minute at 12,000 rpm and thesupernatant was discarded. 50 μl of 1×PBS was added and mixed well. 50μl of 2× loading buffer, also called Sample Buffer or SDS ReducingBuffer was added to the samples and the sample extracts were boiled forexactly 4 minutes. The samples were then immediately loaded ontopolyacrylamide gels (Laemmli, 1970).

Solutions, Standards, and SDS-PAGE Gel

The solutions used in the immunoblot were as follows: (1) 1.5 MTris-HCL, pH 8.8 resolving gel buffer (27.23 g Tris base in 80 ml water,adjusted the pH to 8.8 using 6N HCL and raised the volume to 150 ml. Thesolution was autoclaved and stored at 4° C.) (2) 0.5M Tris-HCl, pH 6.8stacking gel buffer (6.0 g Tris base in 60 ml water, pH to 6.8 using 6NHCl and raised volume to 100 ml. Autoclaved and stored at 4° C.) (3) 10%SDS (10 g Sodium Dodecyl Sulfate and bring up volume to 100 ml withwater and stored at room temperature) (Laemmli, 1970). (4)Acrylamide/Bis solution (from Bio-Rad cat#161-0158). (5) Sample loadingbuffer was an SDS reducing buffer (1.25 ml of 0.5 M Tris-HCl, pH6.8, 2.5ml glycerol, 2.0 ml of 10% SDS and 0.2 ml of 0.5% Bromophenol blue in3.55 ml dH₂O.) 25 μl of β-Mercapto ethanol was added to 475 μl of thesample buffer before use (Sambrook et al., 1989). (6) 10× Electroderunning buffer (30.3 g Tris Base, 144.0 g glycine, 10.0 g SDS and wateradded to bring the volume to 1 L. The buffer was stored at 4° C.) (7)Transfer buffer (300 ml of 10× electrode buffer, 300 ml methanol, 900 mlwater and 0.15 g SDS) (8) 20% APS (200 mg Ammonium persulfate in 1 mlwater) (9) TEMED (N,N,N,N′-Tetra-methyl-ethylene diamine was purchasedfrom BIO-RAD cat#161-0800) (10) 10×PBS (80 g NaCl, 2 g KCl, 26.8 gNa₂HPO₄*7 H₂O, 2.4 g KH₂PO4 and water to a volume of 1 L with pHadjusted to 7.4 with HCl and autoclaved) (Laemmli, 1970).

PEG-Intron (Schering corporation) was used a standard. PEG-Intron iscurrently

FDA approved to be used for Hepatitis C treatment and consists ofrecombinant IFNα2b conjugated to monomethoxy polyethylene glycol. ThePEG portion weighs 12 kDa and IFNα2b weights 19,271 daltons.PEG-Intron's specific activity is 0.7×10⁸ IU/mg protein. Pegylation ofIFNα2b resulted in an increased half-life and lower blood clearancelevels thereby reducing the dosing frequency compared to thenon-pegylated form (Schering Corporation). Dilutions of the standard wasmade from aliquots of 160 μg/ml PEG-Intron stock stored at −4° C. 5 μlof the 160 μl/ml stock was mixed with 95 μl of Peg H₂O and a 8 ng/μlworking stock was made.

All apparatus (glass plates and combs) to be used in the experiment werecleaned using 70% ethanol. Two sets of plates were inserted into theplastic green clamps with the shorter plates to the front. The glassplates were leveled and locked into clamps. The apparatus was placed ina holder with a foam strip on the bottom to form a seal and to preventleakage. The plates were checked for leaks with dH₂O water and it wasblotted out with filter paper. A 15% resolving gel was prepared in a 15ml screw cap tube using 2.4 ml DDI H₂O, 5.0 ml of 30% Bio-Rad degassedBis Acrylamide, 2.5 ml of 1.5 M pH8.8 Tris-HCL gel buffer, and 100 μl of10% SDS. 50 μl of 20% APS and 10 μl TEMED were added to the mixture andswirled to mix. The gel was immediately pipetted into the glass platesleaving room at the top for the stacking to added later. A 0.1% SDSsolution was used to fill the space in the plates on top of the gel tolevel and prevent bubbles. The gel polymerized in 20 minutes and filterpaper was used to remove the 0.1% SDS solution. A 4% stacking gel wasprepared using 6.1 ml DDI H₂O, 1.3 ml Acrylamide/Bis, 2.5 ml of 0.5MTris-HCl, pH6.8 buffer, and 100 ul of 10% SDS. 50 μl of 20% APS and 10μl TEMED were added to the mixture and swirled. The gel was immediatelypipetted over the resolving gel until reaching the top of the glassplates. The 10 well combs were carefully inserted and checked to makesure bubbles were not formed. The gel polymerized in 20 minutes whilethe samples were prepared (Laemmli, 1970).

Equal volumes of the samples and the sample-loading buffer were mixed aswell as the desired concentrations of the standards were prepared andalso mixed with the sample-loading buffer. 15 μl of the protein extractswas used. After the stacking gel polymerized, the combs were removed andthe plates were removed from the casting frame and placed in anelectrode assembly. The assembly was locked and placed in a tank. Thetank was filled with 1× running buffer inside and outside. All thesamples and standards were boiled for 4 minutes and loaded carefullywith a loading tip in to their respective wells. 5 μl of precision plusprotein marker (Bio-Rad) was also loaded into one of the wells. The gelwas run for an hour at 50 V or until the samples were stacked on top ofthe resolving gel and then run for 3-4 hours at 80 V (Sambrook et al.,1989).

Transfer of Protein to Membrane and Immunoblot Analysis

After running the gel the specified time, the glass plates werecarefully separated and the stacking gel portion was removed. A glassdish was used to assemble the transfer apparatus. Transfer buffer waspoured into the dish and the cassette was placed in it. A thin spongewas soaked in transfer buffer and placed on the black side of thecassette. The sponge was topped with a piece of wet filter paper cut tothe same size. The gel was placed into the transfer buffer in the glassdish and carefully removed from the glass plate. The gel was placed ontop of the filter paper and the bubbles were removed. A 0.2 μmTrans-Blot nitrocellulose membrane (Bio-Rad) was moistened and placed ontop of the gel. Then, wet filter paper was placed on top of the membraneand a wet sponge placed on top of the paper. The cassette was closed.The assembly was placed into a mini transfer blot module containing anice pack, a magnet and transfer buffer. The transfer process was run at85V for 1 hour. After the transfer, the membrane was washed with waterand stored overnight at −20° C. The next day, the membrane was removedfrom freezer and incubated in P-T-M (1×PBS, 0.1% Tween 20 and 3% Milk)at room temperature in a shaker for 1.5 hours. During the incubationperiod, a primary antibody solution was made by adding 5 μl of Mousemonoclonal antibody against Human Interferon Alpha (PBL labs 21100-2) to15 ml P-T-M (1:3000 dilution). After the incubation period, the P-T-Mwas discarded from the membrane and the membrane was incubated in theprimary antibody solution for 2 hours at room temperature in the shaker.During the incubation period, a secondary antibody solution containing 5μl of Goat Anti-Mouse IgG conjugated peroxidase (Sigma, St. Louis, Mo.)in 20 ml P-T-M (1:4000 dilution) was prepared. After the incubationperiod, the primary antibody solution was discarded and the membrane wasrinsed with water, two times. The membrane was then incubated for 1.5hours in secondary anotbody solution. After the incubation period, thesecondary antibody solution was discarded and the membrane was washedwith P-T (1×PBS, 0.05% Tween 20) three times for 15 minutes each wash. Afinal wash with 1×PBS was done for 10 minutes. A chemiluminescentsubstrate solution for HRP (Pierce, Rockford, Ill.) was prepared bymixing 750 μl of Luminol Enhancer and 750 μl of stable peroxide in thedarkroom. The chemiluminescent solution was added to the membrane andrinsed over the membrane several times. The chemiluminescent membranewas exposed to an X-ray film in the darkroom and developed in a filmprocessor (Sambrook et al., 1989).

Bombardment of the pLD-BB1 Vector

Generation Media for Tobacco Plants

MSO media was prepared by adding 30 g sucrose and one 4.3 g packet ofMurashige & Skoog (MSO) salt mixture (Gibco BRL) to 1 L dH₂O. Thesolution was mixed well and the pH was adjusted to 5.8 with 1N KOH. 7g/L phytagar was added to a 1 L flask and the mixture was autoclaved.The autoclaved mixture was cooled slightly and poured into Petri dishesand allowed to solidify. The regeneration media of plants (RMOP)solution is prepared exactly like the MSO media with the addition ofgrowth hormones and vitamins (1 ml benzylaminopurine, BAP (1 mg/mlstock); 100 □l napthalene acetic acid, NAA (1 mg/ml stock), 1 mlthiamine hydrochloride (1 mg/ml stock)) to interfere with rootdevelopment; therefore, only shoots would be produced (Daniell, 1993;Daniell, 1997).

Preparation of Microcarriers

In a microcentrifuge tube, 50 mg of gold particles (0.6 μm) were placedand 1 ml of 70% ethanol was added. The mixture was vortexed andincubated at room temperature for 15 minutes. After the incubation, themixture was centrifuged and the gold particles formed a pellet in thebottom of the tube. The supernatant was removed and discarded. Then, 1ml of sterile H₂O was added to the particles and the tube was vortexedagain. After vortexing, the particles were to rest 1 minute and thenwere centrifuged again for 3 seconds. The supernatant was removed anddiscarded. These steps were repeated three times. Then, 50% glycerol wasadded to a concentration of 60 mg/ml and the gold particles were storedat −20° C. 50 μl of gold particles was removed from the stock stored at−20° C. and placed in a microcentrifuge tube. 10 μl plasmid DNA(pLD-smGFP-IFNα5) was added to the gold particles. Then, 50 μl of 2.5MCaCl₂ (prepared that day, 367.5 mg of CaCl₂ into 1 ml of d/aH₂O) wasadded and vortexed. Finally, 0.1M spermidine (20 μl) was added. The tubewas placed in 4° C. and vortexed for 20 minutes. The mixture was thenwashed by adding 200 μl of absolute ethanol to each tube, centrifugingfor 2 s and the ethanol was discarded. The wash was repeated 4 times.Following the wash, the gold particles were resuspended using 30 μl ofabsolute ethanol. The microcentrifuge tubes were then placed on ice.

Microprojectile Bombardment

The macrocarrier holders and stopping screens were autoclaved tosterilize them. The macrocarriers and rupture disks were soaked in 70%ethanol for 15 minutes. The macrocarriers and rupture disks were thenplaced in a sterile Petri disk and allowed to air dry in the hood. Theentire hood and all interior parts of chamber of the gene gun (Bio-RadPDS-1000/He) were cleaned with 70% ethanol to sterilize them. The pumpwas turned on and the main valve of the helium tank was opened. The genegun valve controlling pressure was allowed to reach 13500 psi and set.Stopping screens were placed in macrocarrier holders prior to adding themacrocarrier. Macrocarriers were placed in holders and 6 μl of particlemixture was spread evenly onto the macrocarrier. Five macrocarriers wereused for every tube. The gold suspension was allowed to dry and themacrocarrier holders were placed in the launch assembly with goldparticles facing downwards. One rupture disk was placed in its holderand screwed in place at the top of the vacuum chamber. The secure ringwas screwed onto the launch assembly and the assembly placed in thechamber slot below the rupture disk holder. A piece of sterile whatman#1 filter paper was placed on solidified RMOP media in a petri dish.Each leave clipped from wild-type (untransformed) sterile plants in jarswas taken from the middle of plant and only healthy leaves were choosenof medium size. One leaf at a time was placed on the whatman paperabaxial side upwards because the waxy, thick cuticle lowerstransformation efficiency. The petri dish with leaf was placed on aplastic holder and placed in the next to last slot in the vacuumchamber. The chamber door was closed and secured. The power switch forthe gene gun was turned on. A vacuum was allowed to build to 28 psi inthe bombardment chamber. When 28 psi was reached, the fire switch waspressed until the rupture disk ruptured (˜1100 psi). After delivery ofthe gold particles with vector DNA, the vacuum was released and thepetri dish containing the leaf retrieved. After bombardment, covers wereplaced on petri dishes with the bombarded leaf abaxial side facing upand the dishes were wrapped in aluminum foil and kept in the dark for 48hours to recover from the shock of bombardment.

Results

A 5.9 kb expression vector created by Lee and Daniell contain uniquefeatures facilitating the genetic engineering of plant chloroplasts(FIG. 28). Cloned chloroplast DNA is integrated into the plastid genomethrough site-specific homologous recombination allowing for theexclusion of vector DNA (Kavanagh et al., 1999). This universalchloroplast vector, pLD-CtV, contains the trnI & trnA homologousflanking sequences (chloroplast transfer RNAs coding for isoleucine andalanine) from the inverted repeat region of the chloroplast genome forsite specific integration via homologous recombination (Daniell, 1999).The pLD-CtV also contains the 16S rRNA promoter, the aadA gene encodingspectinomycin resistance (selectable marker), and psbA3′ untranslatedregion to enhance translation. The pLD-BB1 vector contains smGFP genewith a C-terminal fusion of the IFNα5 gene with a furin cleavage sitebetween the fusion proteins cloned into the universal chloroplastvector, pLD-CtV (FIG. 29). Chloroplast integration of the smGFP-IFNα5genes was confirmed by PCR and Southern blot analysis. The smGFP-IFNα5fusion protein expression was confirmed by immunoblot analysis and smGFPexpression under UV light. Expression was quantified by ELISA. ThesmGFP-IFNα5 fusion protein is being further analyzed via in vivostudies. The expression of smGFP-IFNα5 transgenic chloroplasts willfacilitate the provision of a new and alternate treatment for HCV andpossible oral delivery options with a lower cost of production.

Example 5 Evaluation of Chloroplast Derived Cholera Toxin B Subunit(CTB) and Green Fluorescent (GFP) Fusion Protein for Oral Delivery

Many infectious diseases require booster vaccinations or multipleantigens to induce and maintain protective immunity. Advantages ofplant-derived vaccines include the delivery of multiple antigens, lowcost of production, storage & transportation, elimination of medicalpersonnel and sterile injections, heat stability, antigen protectionthrough bioencapsulation, the generation of systemic & mucosal immunityand improved safety via the use of a subunit vaccine and absence ofhuman pathogens. In an effort to study the oral delivery of therapeuticproteins using the transmucosal carrier CTB, a fusion of CTB-smGFP wasexpressed in transgenic chloroplasts of Nicotiana tabacum var. petitHavana by inserting the CTB and smGFP genes into the chloroplast genome.The pLD-CTB-smGFP vector contains CTB with a C-terminal fusion to smGFPseparated by a furin cleavage site. Both genes were inserted into auniversal chloroplast vector, pLD-ctv containing the 16S rRNA promoter,the aadA gene coding for spectinomycin selectable marker gene, the psbA5′ & 3′ untranslated regions to enhance translation in the light andtrnI, trnA homologous flanking sequences for site specific integrationinto the chloroplast genome. Chloroplast integration of the CTB-smGFPgenes was confirmed by PCR and Southern blot analysis. The CTB-smGFPfusion protein expression was confirmed by smGFP expression under UVlight and immunoblot analysis. Expression level was quantified by ELISA.GM1-ganglioside binding assays confirmed that the chloroplast-derivedCTB binds to the intestinal membrane receptor of cholera toxin,confirming correct folding and disulfide bond formation of CTB pentamerswithin transgenic chloroplasts. Functional studies are being carried outin mice to investigate the concept of bioencapsulation by plant cells byusing smGFP as a visible marker as well as to test the ability ofchloroplast-derived CTB to act as a transmucosal carrier of a reportergene product. These investigations might facilitate the development of anovel cost effective oral delivery system for vaccines and therapeuticproteins.

One of the most challenging problems of human health management is thehigh cost of prescription drugs in developed countries and their lack ofavailability in developing countries. For example, interferon (IFN)alpha 2b is used for the treatment of viral diseases such as hepatitisC, as well as for certain cancers. However, IFN treatment for fourmonths costs $26,000 in the United States, where more than forty-fivemillion Americans do not have health insurance (1). Several hundredmillion people in developing countries are infected with hepatitis, butthe daily income of one-third of the world population is less than $2per day (1). The high cost of prescription drugs is due to a numberreasons, including fermentation-based production (each fermenter costsseveral hundred million dollars to build), expensive purification and invitro processing methods (such as column chromatography, disulfide bondformation) (2), the need for storage and transportation at lowtemperature and delivery via sterile injections requiring theinvolvement of hospitals and highly qualified health professionals (1).Therefore, new approaches to minimize or eliminate most of theseexpenses are urgently needed. Transgenic plants offer many advantages,including the feasibility of the oral delivery of foreign proteins, lowcost of production, storage and transportation, heat stability andprotection through bioencapsulation, elimination of the need forexpensive purification, in vitro processing, and sterile injections(1-5). The generation of systemic and mucosal immunity (6) or inductionof oral tolerance (7), improved safety, and absence of human pathogens(3) are other additional advantages (4, 5).

Chloroplast genetic engineering has recently become an attractive methodfor production of recombinant proteins (8, 9) because of highconcentration of transgene expression [up to 47% of the total solubleprotein (10)] due to the presence of 10,000 copies of the transgene percell, which is uniquely advantageous for oral delivery of therapeuticproteins or vaccine antigens. It is also an environmentally friendlyapproach due to effective gene containment offered by maternalinheritance of chloroplast genomes in most crops (11, 12) or engineeredcytoplasmic male sterility (13). Multigene engineering in a singletransformation event (10μ, 14, 15) should facilitate delivery ofpolyvalent vaccines or expression of therapeutic proteins with multiplesubunits.

Despite these advantages, a major limitation remains in the efficientdelivery of plant-expressed therapeutic proteins across the intestinalmucus membrane, primarily because of poor permeability across theintestinal epithelial layer (16). Receptor-mediated oral delivery acrossthe intestine might serve as a possible way to deliver not only vaccinesbut also biopharmaceutical proteins. Ganglioside M1 (GM1) receptors onthe intestinal epithelial cells have been utilized by various pathogenssuch as V. cholerae to facilitate entry of cholera toxin, into theintestine. Crystal structures (17-19) of bacterial toxins like choleratoxin, (CT), heat-labile enterotoxin (LT), and shigella toxin show thatthey belong to AB5 subunit family. In CT, five identical (11.6 kDa)peptides assemble into a highly stable pentameric ring called the Bsubunit (58 kDa). The nontoxic B subunit (CTB) exhibits specific andhigh-affinity binding to the oligosaccharide domain of ganglioside GM1(a lipid-based membrane receptor) and functions to tether the toxin tothe plasma membrane of host cells (17, 20, 21). This receptor is presenton the intestinal epithelium as well as motoneurons and sympatheticpreganglionic neurons (22). GM1 sorts the CT into lipid rafts and aretrograde trafficking pathway to the endoplasmic reticulum, where theenzymatic subunit is transferred to the cytosol, probably by dislocationthrough the transloconsec61P (20).

To test the concept of receptor-mediated oral delivery of foreignproteins, the inventors have constructed a unique cholera toxin B-greenfluorescent protein (CTB-GFP) fusion gene with a furin cleavage sitebetween CTB and GFP and expressed the fusion protein in transgenicchloroplasts. Furin, a member of prohormone-proprotein convertases (23)(PCs), is a ubiquitously expressed protein found in the trans-Golginetwork (TGN) (24, 25), endosomes, plasma membrane, and extracellularspace (26). Furin cleaves protein precursors with narrow specificityfollowing basic Arg-Xaa-Lys/Arg-Arg-like motifs (27). The furin cleavagesite between CTB and GFP would, therefore, facilitate intracellularcleavage of the target protein (GFP).

Transgenic leaves expressing the CTB-GFP or IFNGFP fusion protein werefed to Balb/c mice to investigate receptor-mediated oral delivery offoreign protein using CTB as a transmucosal carrier across theintestinal epithelium. In this study, we show that CTB-GFP binds to theintestinal mucous membrane, including the lymphoid tissue. Experimentalobservations suggest that GFP is cleaved from CTB in the intestinethrough the action of furin and enters the mucosal vasculature. We showthat GFP, but not CTB, is delivered to the liver and spleen of theCTB-GFP fed mice. No significant levels of GFP were observed in theliver and spleen of mice fed with IFN-GFP, which suggests that atransmucosal carrier is essential for efficient delivery of proteinsacross the intestinal lumen. Thus, CTB successfully delivers its fusionprotein to the systemic circulation and supports the use of transmucosalcarriers in the delivery of therapeutic proteins.

Materials and Methods Construction of Chloroplast Vector

The pLD-CTB-GFP construct was based on the universal chloroplast vectorpLD (FIG. 39) that has been used successfully in the inventorslaboratory (28-31). CTB-GFP construct was engineered with a furincleavage site, Pro-Arg-Ala-Arg-Arg, in between CTB and GFP. Theconstitutive 16 s rRNA promoter was used to drive transcription of theaadA and the CTB-GFP genes. The aminoglycoside 3_adenylyltransferase(aadA) gene conferring spectinomycin resistance was used as a selectablemarker. The 5_-UTR from psbA, including its promoter, was engineered toenhance translation of the CTB-GFP because it has several ribosomalbinding sites. The 3′UTR region conferred transcript stability. AGFP-IFN alpha5 fusion construct with a furin cleavage site between thetwo genes was created and expressed in Nicotiana tabacam chloroplasts,which served as a control molecule for the delivery of GFP without atransmucosal carrier.

Bombardment and Selection of Transgenic Plants

The Bio-Rad PDS-1000/He biolistic device was used to bombard pLD-CTB-GFPonto sterile Nicotiana tabacum cv. Petit Havana tobacco leaves, on theabaxial side as has been described previously (29, 30, 32). Thebombarded leaves were incubated in the dark for 24 h and then placed onshooting media (RMOP) containing 500 μg/ml spectinomycin for two roundsof selection.

Pcr Analysis to Test Stable Integration

DNA was isolated from the transgenic shoots by using Qiagen DNeasy PlantMini Kit, and PCR analysis was performed to confirm integration of thetransgene in the inverted repeat regions of the chloroplast genome. PCRreactions were performed with two sets of primers, 3P/3M and 5P/2M (28).

The samples were denaturated for 5 min at 95° C. followed by 30 cyclesof the following temperatures: 95° C. for 1 min, 65° C. for 1 min, and72° C. for 2 min and a 72° C. hold for 10 min after all 30 cycles werecompleted. After confirmation of transgenic plants, the shoots were thentransferred to a rooting medium (MSO) with 500 μg/ml spectinomycin as aselective agent.

Southern Blot Analysis

Total plant DNA was digested with EcoR1, separated on a 0.7% agarose gelat 45V for 8 h, and then transferred to a nitrocellulose membrane.pUC-computed tomography vector DNA was digested with BamHI and BglII togenerate a 0.8 kb probe, which was used as a flanking probe (28). Afterlabeling the probe with P32, hybridization of the membranes wasperformed by using Stratagene QUICK-HYB hybridization solution andprotocol (Stratagene, La Jolla, Calif.).

Western Blot Analysis

Approximately 100 mg of leaf tissue was ground in liquid nitrogen andresuspended in 500 μl of plant extraction buffer (0.1% SDS; 100 mM NaCl;200 mM Tris-HCl, pH 8.0; 0.05% Tween 20; 400 mM sucrose; 2 mM PMSF).After centrifugation at 13,000 rpm for 5 min, the supernatant containingthe extracted protein was collected. We boiled 10 μl of the plantextract along with 10 μl of sample loading buffer, which was then run ona 15% SDS-PAGE gel for 40 min at 50 V and then 2 h at 80 V. The proteinwas then transferred to nitrocellulose membrane for 1 h at 80 V. Afterblocking the membranes with PTM (1×PBS, 0.05% Tween 20, and 3% dry milk)for 1 h, we added polyclonal rabbit anti-CTB primary antibody (Ab)(Sigma) 1:3000 dilution. Goat anti-rabbit IgG conjugated to alkalinephosphatase (Sigma) at a 1:5000 dilution was used as a secondary Ab.

Furin Cleavage Assay

Approximately 100 mg of leaf material was powdered in liquid nitrogenand resuspended in 500 μl of plant extraction buffer containing 15 mMNa2CO3, 35 mM NaHCO3, 3 Mm NaN3, 5 mM CaCl2, and 0.5% Triton-X,2-mercaptoethanol at pH 6.0 and 7.0. We added 1 mM PMSF to some of thesamples. After centrifugation at 13,000 rpm for 5 min, the supernatantcontaining the extracted protein was collected.

The extract (20 μl) was incubated at 30° C. for 4 h with 4 U of furin. Acontrol group was also incubated at 30° C. for 4 h without furin. After4 h, each sample was mixed with 20 μl sample loading buffer, boiled, andrun on 12% SDS-PAGE gel for 45 min at 80 V and then 2 h at 100 V. TheWestern blot analysis was performed as per the procedure outlined above.Chicken anti-GFP Ab (Chemicon) at a 1:3000 dilution was used as theprimary Ab, and alkaline phosphatase conjugated rabbit antichicken IgG(Chemicon) at a dilution of 1:5000 was used as a secondary Ab.

ELISA

The CTB-GFP quantification was done using the ELISA (ELISA). Thestandards and test samples were diluted in coating buffer (15 mM Na2CO3,35 mM NaHCO3, 3 mM NaN3, pH 9.6). The standards, ranging from 50 to 500ng, were made by diluting recombinant GFP in 1% PBS. The leaf sampleswere collected from plants exposed to regular lighting pattern (16 hlight and 8 h dark), and total protein was extracted using plant proteinextraction buffer. Standard GFP dilutions (100 μl) and protein sampleswere bound to a 96-well plate overnight at 4° C. The background wasblocked with fat-free milk in PBST for 1 h at 37° C. followed by washingwith PBST and water. Primary Ab used was polyclonal chicken anti-GFP Ab(Chemicon) diluted (1:3000) in PBST containing milk powder. Secondary Abwas HRP-conjugated rabbit anti-chicken IgG-secondary Ab (Chemicon) at a1: 5000 dilution in PBST containing milk powder. For the color reaction,100 μl of 3,3_(—),5,5_-tetramethyl benzidine (TMB from American Qualex)substrate was loaded in the wells and incubated for 10-15 min at roomtemperature. The reaction was stopped by addition of 50 μl of 2Nsulfuric acid per well, and the plate was read on a plate reader (DynexTechnologies) at 450 nM.

GM1 Binding Assay

To test the functionality of CTB-GFP expressed in chloroplasts, aCTB-GM1 binding assay was performed. We coated 96-well plates with 100μl of monosialoganglioside-GM1 (Sigma) (3.0 ng/ml in bicarbonate buffer)and incubated them overnight at 4° C. After washing with PBST and water,the standards and samples were incubated for 1 h at 37° C. The plate wasblocked with 1% BSA in 1×PBS for 1 h at 37° C. Rabbit anti-CTB primaryAb (Sigma) and alkaline phosphatase (activating protein) conjugated goatanti-rabbit secondary Ab (Sigma) was used to detect the CTB binding toGM1 receptor. The plates were washed with PBST and water, and 200 μl ofthe substrate p-Nitrophenyl phosphate (PNPP) was added to the wells andincubated in the dark at 37° C. for 20 min The reaction was stopped byadding 50 μl of 3N NaOH, and the plates were read on a plate reader(Dynex Technologies) at 405 nM.

Animal Studies

Three groups of 5-week-old female Balb/c mice were fed with CTB-GFP, IFNalpha5-GFP (IFN-GFP), and wild-type (untransformed) plant leaf material.Leaves (350 mg) were powdered in liquid nitrogen, mixed with peanutbutter, and fed to the mice, which had been starved overnight prior tothis experiment. The mice were then gavaged for two more days, two timesa day, with 40 mg of leaf material per gavage that was powdered withliquid nitrogen and mixed with 0.1M PBS (PBS). Five hours after the lastgavage, the mice were sacrificed and perfused with 10 ml of PBS followedby 4% paraformaldehyde in PBS. Fresh frozen sections of the liver,spleen, ileum, and jejunum were collected according to Samsam et. al(33). Additional tissue was removed and immersed in Tissue Tec freezingmedium (Vector labs) and immediately frozen in nitrogen-cooledisomethylbuthane (Sigma). Fixed tissue was cryoprotected by passingthrough 10, 20, and 30% sucrose solutions in PBS. Frozen sections (10 _mthick) of various tissues were then made using a cryostat.

Fluorescence Microscopy and Immunohistochemistry for GFP, CTB, andImmune Cells

Frozen sections (10 _m thick) of intestine, liver, and spleen weremounted with PBS and observed for GFP fluorescence using a Leica 4500microscope Immunohistochemistry was performed in order to show thepresence of GFP and/or CTB in various tissues. The slides were firstblocked with 10% BSA (BSA) and 0.3% Triton-X 100. Polyclonal chickenanti-GFP (Chemicon) or polyclonal rabbit anti-CTB (Sigma) primaryantibodies, at a concentration of 1:500 and 1:300, respectively, in 1%BSA and 0.3% Triton-X, were used for GFP or CTB localization of thetissues. Those sections processed for HRP conjugated secondaryantibodies were blocked with a mixture of methanol/hydrogen peroxide 30%(2:1 ratio) to block the endogenous peroxidases. The secondaryantibodies were horseradish peroxidase (HRP)-conjugated rabbitantichicken IgG (Chemicon) or HRP-conjugated goat anti-rabbit (Sigma).Tissue-bound peroxidase was developed by using the 3,3_diaminobenzidine(3,3_-diaminobenzidine) as a substrate to visualize the immunoreaction.

For macrophage localization of the tissues, rat monoclonal F4/80 Ab(Serotec) was used according to Berghoff et al. (34). The secondary Abwas Alexa-555 conjugated Goat antirat IgG (Molecular Probes). Americanhamster anti-CD11c primary Ab and anti-hamster Alexa-546 conjugatedsecondary Ab (Molecular Probes) were used to visualize dendritic cellsin the intestine and other tissues. FITC-labeled anti-chicken IgG wasused as a secondary Ab in such immunofluorescence staining to detect GFPin tissues.

Results

Confirmation of transgene integration into chloroplast genome

Nicotiana tabacum cv. Petit Havana leaves were bombarded with thepLD-CTB-GFP vector, and the leaves were grown on selective mediumcontaining 500 mg/l spectinomycin. The resultant shoots were thenscreened for chloroplast transformants by PCR analysis by using primers3P/3M, and 5P/2M (FIG. 39A-C). The 3P primer lands on the nativechloroplast genome upstream of the site of integration, whereas the 3Mprimer lands on the aadA transgene producing a 1.65 kb PCR product. Thisanalysis ruled out the nuclear transformants because 3P primer would notanneal and the spontaneous mutants are eliminated because 3M primerwould not anneal.

To check for the presence of the transgene in the chloroplast, weperformed the 5P-2M PCR analysis. The 5P primer lands on aadA gene andthe 2M lands on the trnA coding sequence, which produces a 2.9 kb PCRproduct with CTB-GFP. This confirmed the site-specific integration ofthe CTB-GFP fusion gene in the inverted repeat regions of thechloroplast genome.

Southern Blot Analysis to Investigate Homoplasmy

To further confirm the integration of the transgene into the chloroplastgenome and to determine whether homoplasmy had been achieved, Southernblot analysis was performed. Total plant DNA was digested with theenzyme EcoR1 and hybridized with a chloroplast flanking sequence probe(0.8 kb). Wild-type plants generated a 4.4 kb fragment, and transgenicplants generated 4.9 and a 2.2 kb fragments (FIG. 39D). All of thetransgenic lines tested appeared to be homoplasmic (within the levels ofdetection), which means that all of the chloroplast genomes within plantcells contained the transgene CTB-GFP.

GFP Expression and Assembly of CTB-GFP Pentamers in Transgenic Lines

FIG. 40 shows the transgenic and wild-type (WT) plants. In FIG. 40B, theGFP expression of the transgenic plants can be seen under the UV light,which is not seen in the wild-type (untransformed) plant (FIG. 40A).FIG. 40C shows WT plant, and FIG. 40D, the CTB-GFP expressing plantunder a low-magnification microscope. Expression of GFP is clearlyevident in FIG. 40D. Western blot analysis was performed to investigatethe expression of the fusion protein CTB-GFP in transgenic tobaccochloroplasts (FIG. 41A). The pentameric form (188 kDa) was observed inthe unboiled samples of the transgenic plants, while predominantly themonomeric form (37.6 kDa) was detected in boiled samples.

Furin Cleavage Assay

The protease furin is present in the constitutive secretory pathway andon the cell surface of virtually all cells (35). An in vitro furincleavage assay was performed on the CTB-GFP expressing plant extract toshow that the engineered cleavage site (Arg-Ala-Arg-Arg) was recognizedby furin. As seen in FIG. 41B, a 26 kDa polypeptide that correspondedwith the recombinant GFP protein was observed in the samples that wereincubated with furin, thus proving that furin could cleave CTB-GFP torelease GFP. Furin cleavage occurred at both pH 6.0 and 7.0 in thesamples with and without PMSF. Still, some protein did not get cleaved,probably because the amount of enzyme was not sufficient to cleave allthe CTB-GFP protein present in the plant extract. The incubation time of4 h might also have been insufficient. However, the presence of thecleaved GFP product in the samples incubated with furin confirms thatthe engineered furin cleavage site is functional. The introduction offurin consensus sequences at the Bchain/C-peptide and theC-peptide/A-chain interfaces of human proinsulin has been demonstratedto increase the processing of proinsulin to mature insulin in a widevariety of non-neuroendocrine cells, including fibroblasts, myoblasts,epithelial cells, and lymphocytes (36-42). As the furin cleavage site isalso recognized by the endopeptidases PC2 and PC3/1, it is likely thatCTB-GFP fusion protein is cleaved more efficiently during the process ofreceptor-mediated delivery.

Quantification of CTB-GFP

To quantify the amount of CTB-GFP fusion protein in transgenic tobaccoleaves, ELISA (ELISA) was performed (FIG. 41B). A standard curve wasobtained using different concentrations of recombinant GFP. The amountof CTB-GFP in the transgenic plants was compared with the knownconcentrations of the recombinant GFP (standard curve). Expressionlevels of CTBGFP ranged from 19.09 to 21.3% total soluble protein.

GM1 Binding Assay

The functionality of chloroplast-derived CTB-GFP was determined by itsability to bind to GM1 in an in vitro GM1 binding assay (FIG. 41C). GM1binding assay showed that pentamers of CTB-GFP were formed. This findingconfirms the correct folding and disulfide bond formation of CTBpentamers within transgenic chloroplasts because only the pentamericform of CTB can bind to GM1 (21).

Fluorescent Microscopy to Detect the Presence of GFP in the Tissue

Fixed tissue and fresh frozen sections of the liver, spleen, ileum, andjejunum were made from the three groups of mice fed with plantsexpressing CTB-GFP, IFN-GFP, and WT plants, respectively. In mice fedwith CTB-GFP expressing plant leaf material, fluorescence microscopyshowed the presence of GFP in intestinal mucosa and submucosa (FIG.42A), the hepatocytes of the liver (FIG. 42D) as well as various cellsof the spleen (FIG. 42G). In the mice fed with wild-type (untransformed)leaf material, no GFP fluorescence was observed (FIGS. 42B, E, and H).In the mice fed with IFN-GFP expressing plant leaf material, no GFP wasdetected in the liver or spleen (FIGS. 42F and I). Detection of GFP inthe liver and spleen following oral delivery of CTB-GFP expressing plantleaf material, suggests the successful delivery of the protein acrossthe intestinal lumen into the systemic circulation. Moreover, the lackof detection of a significant amount of GFP in the liver and spleen ofmice fed with IFN-GFP expressing plants suggests that a transmucosalcarrier such as CTB is required for delivery of an adequate amount of amacromolecule across the intestinal lumen into the systemic circulation.

Immunohistochemistry

To confirm the fluorescent microscopy findings, immunostaining wasperformed with both CTB and GFP antibodies. In the intestine of the micefed with CTBGFP, anti-GFP Ab detected GFP inside the epithelial cells ofthe villi of the intestine, in the crypts, as well as in the submucosaltissue (FIG. 43 A, C), which suggesting GFP uptake by lymphoid cells aswell as the circulation. These results confirmed the previous microscopyfindings (FIG. 42) and showed the presence of GFP in various tissues,confirming that GFP was successfully delivered to blood when transgenicleaf material was orally fed to the mouse. GFP immunoreactivity wasdetected in the liver and spleen (FIGS. 43E and H) in a similar patternto that seen with fluorescence microscopy of the native tissue (FIGS.42D and G). In the case of the mice fed with wild-type leaf material, noGFP was detected in any of the tissues (FIGS. 43F and I). In the micefed with plants expressing IFN-GFP, GFP was not detected in the liver orspleen cells (FIGS. 43G and J).

To study the route of CTB in the body, we performed immunohistochemistryusing anti-CTB antibodies. CTB was detected in the intestinal cells aswell as inside the villi (FIG. 44A) in the lamina propia and thesubmucosa. It was, however, not detected in the liver (FIG. 44E),indicating that GFP is cleaved away from CTB and that, while GFP leavesthe cell, CTB probably is translocated to the basolateral membrane ofthe cell. These results support the feasibility of CTB to act as atransmucosal carrier and orally deliver fused proteins via theintestinal cells. To localize the GFP and/or CTB in the gut associatedlymphoid tissue (GALT) and other tissues, double staining forantigen-presenting cells such as macrophages or dendritic cells wasperformed. A double staining with F4/80 Ab for macrophages showed thepresence of CTB inside macrophages (FIG. 44C). FIG. 44G showsmacrophages associated with GFP, and FIG. 44I shows dendritic cellstaking up the GFP. In either case, associations of GFP with theseantigen presenting cells were found. Most of the macrophages were notassociated with GFP, which is perhaps due to uptake by the blood andlymph circulation, while the CTB is translocated to the basolateralmembrane and is associated with macrophages.

Discussion

In this study, detection of GFP and CTB in the intestinal mucosa (FIGS.43, 44) suggests that CTB-GFP has been taken up by the enterocytes andthe gut-associated lymphoid tissue (GALT). The CTB domain of the CTB-GFPforms the pentameric structure within chloroplasts through disulfidebond formation; pentameric form binds to the GM1 receptors onenterocytes and is endocytosed into the intestinal cells as endosomes(20). GM1 functions to concentrate CTB in detergent-insoluble,glycolipid-rich apical membrane microdomains called lipid rafts (43,44). Binding to lipid rafts is required to couple the lipid-anchoredprotein with intracellular machinery for protein sorting and vesiculartraffic (45, 46). After endocytosis, the CTB-GM1 complex traffickingoccurs retrogradely through Golgi cisternae and/or TGN (20, 47) into thelumen of the endoplasmic reticulum (ER; 48). The GM1-CTB-GFP complex inthe lipid rafts, targeted to the TGN, loses its endosomal covering.Within the TGN, ubiquitously expressed furin cleaves numerouspolypeptide precursors as it gets activated. In eukaryotes, manyessential secreted proteins and peptide hormones, enzymes, andneuropeptides are initially synthesized as proproteins (inactiveprecursors) and are activated by proteolytic cleavage by furin and othermembers of the prohormone-proprotein convertase (PCs, 23). Abundantexperimental evidence indicates that the CTB-GFP protein with furincleavage site in between the fusion protein gets cleaved and, as aresult, the CTB and GFP separate. The CTB is taken into the ER and fromthere to the baso-lateral surface of the cell (transcytosis), where itremains membrane bound to GM1 receptor (20). The GFP molecule gettingout of the TGN (presumeably membrane-bound) is exocytosed through thebasolateral membrane and finds its way into extracellular fluid and intothe submucosal vessels, including the lymphatic system. Due to thelarge-size fenestrations of the lymphatic vessels, lymphatics returnover 3 L of fluid and _(—)120 g of protein to the bloodstream every 24 hin an adult human (49).

Besides the entry of CTB-GFP through the GM1 ganglioside receptor, the Mcells in intestinal epithelium covering the mucosa-associated lymphoidtissue in the digestive tract also serve as a port of entry ofmacromolecules and microorganisms by pinocytosis (50). Therefore, asmall amount of CTB-GFP could be taken up by the GALT. This is shown inour study by CTB and GFP expression in the antigen presenting cells,including the macrophages as well as the dendritic cells in theintestinal lamina propia and submucosa Similarly, a small amount of GFPassociated with macrophages in the intestine of the INFGFP fed mice islikely to be taken up by the M cells nonspecifically. The IFN-GFP fusionprotein also contains a furin cleavage site but, due to limited uptakeby the intestinal epithelial cells, there is not a significant GFPtransport to the tissues of the IFNGFP fed mice. The amount of CTB-GFPreaching the enterocytes via GM1 receptor is very high compared with theentry of IFN-GFP through M cells. This is quite evident due to the GFPdetected in various organs of the CTB-GFP fed mice (FIGS. 43, 44).Presence of GFP and not CTB in the liver of CTB-GFP treated mice in ourstudy (FIGS. 43, 44) suggests the cleavage of the CTB-GFP fusion proteinin enterocytes and uptake of GFP into the vasculature of the laminapropia and the submucosa. CTB, however, might be translocated to thebasolateral cell membrane and remain bound to GM1 (20).

The main goal of this study is to develop an efficient oral delivery ofprotein through GM1 receptor-mediated endocytosis. Moreover, furincleavage site facilitates the cleavage of the candidate protein in thecell, so that it could be passed into the extracellular space and intothe circulation. Internalization of GFP using receptor-mediatedendocytosis suggests a possible way of protein delivery across theimpermeable intestinal mucous membrane. Because of the rapid turnover ofthe intestinal epithelial cells (51) in humans (renewal of theintestinal epithelium occurs in every 3-6 d), repeated feeding of theCTB fused to a therapeutic protein is possible due to the continuousavailability of GM1 receptors in the new epithelium. Moreover, Petersonand colleagues suggested a recycling mechanism for GM1 receptor as well(52).

One of the most challenging problems of human health management is thehigh cost of prescription drugs in developed countries and their lack ofavailability in developing countries. Such high cost of therapeuticproteins can be attributed to their production in fermentation-basedsystem, expensive purification and processing methods, low-temperaturestorage, transportation, and sterile delivery using syringes throughhealth professionals. Most of these expenses could be avoided byexpressing therapeutic proteins in plant cells and through their oraldelivery. This study shows internalization of CTB-GFP by the mouseintestinal mucosal cells as well as the antigen-presenting cells in theintestinal mucosa and submucosa. We also show the presence of GFP butnot CTB in the liver of mice following oral delivery of CTB-GFP leafmaterial. Detection of both CTB and GFP in mouse intestinal cellsfollowing oral administration of CTB-GFP expressing leaf material showsthat the recombinant protein has been protected from peptidases and/oracids by bioencapsulation (53) within the plant cells. Several vaccineantigens (28, 54-57) and human blood proteins (31, 58-60) have beenexpressed in transgenic chloroplasts and shown to be fully functional.The ability to express high levels of foreign proteins in plastidspresent within edible plant parts (61, 62) and the rapid turnover ofintestinal epithelial cells (51) for recycling GM1 receptors make thisapproach a reality. This study facilitates the provision of low-costproduction and delivery of human therapeutic proteins.

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Example 6 Characterization of Heterologous Multigene Operons inTransgenic Chloroplasts: Transcription, Processing And TranslationIntroduction

Plastid genes in higher plants are mainly organized as operons, of whichmore than sixty have been described in the tobacco chloroplast genome(Sugita and Sugiura, 1996). These may group genes of related orunrelated functions, the former being the most common (Barkan, 1988;Rochaix, 1996). Most of these genes are transcribed into polycistronicprecursors that may be later processed and modified to render thetranscripts competent for translation (Eibl et al., 1999; Barkan &Goldschmidt-Clermont, 2000; Monde et al., 2000b).

The processing mechanisms for translation regulation in chloroplastgenes of higher plants are still largely unknown. The general consensusis that most native primary transcripts require processing in order tobe functional (Barkan, 1988; Zerges, 2000; Meierhoff et al., 2003), andthat post-transcriptional RNA processing of primary transcriptsrepresents an important control of chloroplast gene expression(Hashimoto et al., 2003; Nickelsen, 2003). However, it is believed thatmore than one pathway may be involved in transcript processing (Danon,1997; Choquet and Wollman, 2002).

For example, several studies have shown that the regulation of geneexpression in the chloroplast relies more on RNA stability than ontranscriptional regulation (Deng and Gruissem, 1987; Jiao et al., 2004).In chloroplast, such stability is mainly influenced by the presence of5′ untranslated regions, or UTRs (Eibl et al., 1999; Zou et al., 2003),nucleus-encoded factors (Lezhneva and Meurer, 2004) and 3′UTRs (Adamsand Stern, 1990; Chen and Stern, 1991), without which rapid degradationor low accumulation of primary transcripts has been observed. The roleof plastid 3′UTRs differs from the role of its bacterial counterparts bybeing more involved in transcript stability and less involved in theeffective termination of transcription (Stern and Gruissem, 1987).

Translation has also been a crucial step in the regulation of geneexpression, as in many cases protein levels in the chloroplast did notcorrelate with steady-state transcript abundance (Monde et al., 2000b).Therefore, the transcription of native chloroplast operons and theirpost-transcriptional and translational patterns have been the target ofseveral studies which showed that intercistronic processing enhancedtranslation of chloroplast operons, including the maize psbB and petclusters (Barkan, 1988; Barkan et al., 1994). In addition, differentspecies may experience various processing mechanisms for the same genecluster. For example, species such as Arabidopsis (Meierhoff et al.,2003), tobacco (Monde et al., 2000a) and spinach (Westhoff and Herrmann,1988) have a different mechanism than maize for the translation of petD,which depends mainly upon the establishment of dicistrons andtricistrons of this gene. Alternative processing of the polycistroncontaining the petD gene, which produces monocistronic petD, causes thedegradation of the transcript, inhibiting translation (Meierhoff et al.,2003; Tanaka et al., 1987; Monde et al., 2000a, b). In contrast, inChlamydomonas, nearly all chloroplast genes appear to be transcribed asmonocistronic mRNAs, with translation being an essential regulatory stepof gene expression (Rochaix et al., 1989; Zerges and Rochaix, 1994).Other mechanisms, such as editing, which can produce alternate startcodons, have been linked to alternative processing and to a completedifferent translation pattern (Hirose and Sugiura, 1997; del Campo etal., 2002). These examples provide evidence of different modificationsof primary transcripts for efficient translation in chloroplasts.

Traditionally, plant genetic engineering had involved the introductionof single genes through nuclear transformation. In the past decade, theintroduction of multiple genes has also been successful through thisapproach, allowing the incorporation of complete metabolic pathways (Maet al., 1995; Nawrath et al., 1994; Ye et al., 2000). However, thisapproach required a long process of integration of individual transgenesfollowed by breeding to reconstruct the desired pathways. Additionally,transgene segregation from nuclear transformed plants may be possible insubsequent generations, which may result in loss of function of theintroduced pathway. Furthermore, plant nuclear genes are typicallytranscribed monocistronically, which requires separate promotersequences for each of the introduced genes. Expression of foreign genesmay also be influenced by position effects and gene silencing, causinglevels of gene expression to vary among independent transgenic lines(Daniell and Dhingra, 2002).

On the other hand, plant genetic engineering through chloroplasttransformation presents several additional advantages over nucleartransformation, such as their ability to efficiently transcribe andtranslate operons (DeCosa et al., 2001; Lossl et al., 2003; Ruiz et al.,2003), as well as to confer hyperexpression capability (Daniell et al.,2004c). In addition, chloroplasts are able to accumulate foreignproteins that are toxic in the cytoplasm, such as cholera toxin □subunit (Daniell et al., 2001), trehalose (Lee et al., 2003), andxylanase (Leelavathi et al., 2003), without any deleterious effects, dueto the compartmentalization of transgene products (Bogorad, 2000).Concerns about position effect are also eliminated due to site-specificintegration of transgenes via homologous recombination of chloroplastDNA flanking sequences (Daniell et al., 2002), and because chloroplastsare maternally inherited in most crops, the risk of outcrossingtransgenes to related species through pollen is minimized (Daniell,2002). Additionally, transformation of plastids in non-green tissues,such as carrot roots, offer promising options for oral delivery ofvaccine antigens (Kumar et al., 2004a).

As foreign genes are engineered into operons, the resulting transcriptdiffers from the native operons by lacking native intergenic sequences.These sequences are removed during cloning or by PCR amplification ofthe coding sequences. The effect of such modifications in thetranscription and translation of heterologous operons has not yet beeninvestigated. Therefore, the purpose of this study is to examine thetranscription, processing and translation of several foreign operonsengineered via the chloroplast genome. The results of this investigationprovide sufficient evidence that suggests that engineered polycistronsin chloroplast transgenic lines are efficiently translated and thatprocessing into monocistrons is not required to obtain overexpression oftransgenes. Additionally, the role of 5′UTRs and 3′UTRs inpost-transcriptional modifications, translation, and transcriptstability are addressed. Addressing questions on polycistron translationas well as the sequences required for processing and transcriptstability are essential for chloroplast metabolic engineering.

Results Multigene Engineering via the Tobacco Chloroplast Genome

Multigene engineering via the chloroplast genome has been achieved byusing several different foreign genes, promoters, and 5′ and 3′regulatory sequences (Daniell et al., 2004a; Kumar and Daniell, 2004).Chloroplast transgenic lines analyzed in this study were geneticallyengineered with multigene cassettes that contained the following basicfeatures; the aadA (aminoglicoside 3′-adenylyltransferase) gene, whichconfers resistance to spectinomycin and help in transgenic plantselection (Goldschmidt-Clermont, 1991), downstream from the constitutivechloroplast 16S ribosomal RNA gene promoter (Prrn). The heterologousgene or genes of interest were inserted downstream of the aadA gene andwere flanked at the 3′ end by the psbA 3′ untranslated region (3′UTR),which is involved in mRNA abundance and stability in the chloroplast(Deng and Gruissem, 1987; Stern and Gruissem, 1987). In some cases, theheterologous gene was also engineered to contain the psbA promoter and5′ regulatory sequence (5′ untranslated region; 5′UTR) to enhancetranslation (Eibl et al., 1999; Fernandez-San Millan et al., 2003;Dhingra et al., 2004, Watson et al., 2004). The multigene cassettes wereflanked at the 5′ and 3′ by sequences homologous to the tobaccochloroplast trnI (tRNA Ile) and trnA (tRNA Ala) genes, respectively,which allow site-specific integration by homologous recombination intothe inverted repeat region of the chloroplast genome (Daniell et al.,1998). More than thirty genes have been successfully integrated andexpressed at this transcriptionally active spacer region (Daniell etal., 2004a, b). In this study, the following foreign genes were insertedinto the basic expression cassettes: human serum albumin (hsa), choleratoxin β subunit (ctxB), ctxB-gfp (green fluorescent protein) fusion,Bacillus thuringiensis insecticidal protein (cry2Aa2) along with theassociated chaperonin protein (orf2) and orf1, and trehalose phosphatesynthase (tps1). The transgenic lines engineered to express CRYinsecticidal protein contained the entire cry2Aa2 native operon.

Transcription and Translation of the cry2Aa2 Operon

The chloroplast transgenic lines transformed with the transgene cassettecontaining the aadA gene and the complete cry2Aa2 operon (ORF1,2-Cry2Aa2lines) were used to study the transcriptional and translational patternsof a heterologous operon in transgenic chloroplasts. This operoncomprises the orf1, orf2, and cry2Aa2 genes under the transcriptionalregulation of the Prrn promoter (FIG. 45A). Several transcripts wereanticipated based on transcription initiation at the engineered promoter(Prrn promoter) and the native 16S ribosomal RNA promoter (native Prrn)in transgenic lines (FIG. 45A). Northern blot analyses of threeindependent lines harboring the cry2Aa2 operon revealed that thepredicted 4.9 kilonucleotide (knt) polycistron, which contained all fourtransgenes (aadA-orf1-orf2-cry2Aa2), was the most abundant transcriptdetected with the cry2Aa2 specific probe (FIG. 45B, c). Interestingly,the cry specific probe also revealed a shorter transcript of about 2.4knt; which was about the same size as the cry2Aa2 gene (FIG. 45B, a),suggesting that this transcript could be the cry2aA2 monocistron.Densitometric analyses of the foreign mRNA transcripts revealed that thecry2Aa2 monocistron and the aadA-orf1-orf2-cry2Aa2 polycistron hadsimilar abundances (FIG. 45C, a,c), indicating that processing in theintergenic region between orf2 and cry2Aa2 occurred in about 50 percentof the polycistrons transcribed from the Prrn promoter (FIGS. 45A, B a,c). Another prominent 7.4 knt transcript was predicted, based on thecalculation of the length of the coding sequence, initiating at thenative 16S Prrn promoter (FIG. 45B, e). A low intensity ˜6.0 knttranscript detected (FIG. 45B, d) may be produced by read-through of thetranscript starting at the Prrn promoter and terminating downstream ofthe engineered 3′ UTR. The low intensity ˜3.5 knt transcript (FIG. 45B,b) terminates at the same location as the 6.0 knt transcript, althoughit is smaller due to the processing between orf2 and cry2Aa2. Becausethis fragment only contained the cry gene and the sequences downstreamfrom the 3′UTR, it could not be detected with the aadA probe (FIG. 45D).The read-through transcripts processed downstream of the 3′UTR representan average of 27.3±3% of those produced in these transgenic lines (FIG.45B, b, d).

Northern blot analyses with the aadA specific probe confirmed theresults observed with the cry2Aa2 probe. The predicted 4.9 kntpolycistron that harbors the aadA gene plus the complete cry operon wasdetected as expected (FIG. 45D, c). Although the predicted 2.5 knttricistron (FIG. 45D, f) containing the aadA gene plus the orf1 and 2was expected due to processing between the orf2 and the cry2Aa2 genes, atranscript of a similar intensity to that of the polycistron wasobserved instead (FIG. 45D, c, f). Densitometric analyses revealed a 1to 1 ratio of the polycistron (aadA-orf1-orf2-cry2Aa2) versus theaadA-orf1-orf2 tricistron (FIG. 45E, c,f), due to processing in theintergenic region between orf2 and cry2Aa2 (FIG. 45A). These resultsshowed that the two transcripts produced by the processing in theintergenic region between cry2Aa2 and orf2 resulted in transcripts witha similar abundance to the complete polycistron containing all fourgenes and the 3′UTR (FIG. 45C, a,c and FIG. 45E, f and c, respectively).The fact that the tricistron containing the aadA, orf1 and orf2 genesdid not contain a chloroplast 3′UTR but still was very stable, suggeststhat polycistrons are stable in the chloroplast even in the absence of3′UTRs. The results obtained by using the orf1-orf2 fragment (orf1,2) asa probe, confirmed the detection of the aadA-orf1-orf2 tricistron(predicted 2.5 knt), indicating effective processing at the intergenicregion between orf2 and cry2Aa2 (FIG. 45F, f). Other transcripts oflarger size were also observed and corresponded to those obtained withthe cry2Aa2 gene-specific probe (FIG. 45 F, d,e).

Northern blots were also performed on the cry2Aa2 transgenic lines usingthe psbA 3′UTR probe (FIG. 51B). Results revealed a pattern similar tothat obtained with the cry2Aa2 gene-specific probe, as well as thepresence of the endogenous psbA transcript. In the case of the cry2Aa2operon, transcripts were in much lower proportion to the native psbAoperon. Because the native psbA and the heterologous cry2Aa2 operon aredriven by different promoters, transcript abundance cannot bequantitatively compared. In contrast to the results obtained in FIG.45B, only the major transcripts (a and c) were detected.

Polysome fractionation assays of the cry2Aa2 operon support polycistrontranslation, as the larger transcripts corresponding in size to thecomplete operon (from the prrn promoter) were observed mainly in thelower fractions of the sucrose gradients, when hybridized with thecry2Aa2 probe (FIG. 46A) and with the orf1,2 probe (FIG. 46B).Additionally, smaller transcripts corresponding in size to the cry2Aa2gene processed from the rest of the operon also appear associated topolysomes, suggesting that processing may also occur and could becoupled to translation. Puromycin release controls confirm that thepolycistronic transcripts found in the lower fractions were indeedassociated to polyribosomes (FIG. 46C).

Western blot analyses revealed that the Cry2Aa2 (65 kDa; F1G. 45H) andthe ORF2 proteins (45 kDa; FIG. 45) were highly expressed in thetransgenic lines. The abundant expression of the orf2 confirmed thatpolycistrons were efficiently translated without the need for processinginto monocistrons.

Transcription and Translation of the hsa Operon

Chloroplast transgenic lines transformed with 3 different multigeneconstructs, all containing the human serum albumin (hsa) gene, were usedto study transcription, translation and posttranscriptionalmodifications (FIG. 47A). The Prrn promoter drives the operon downstreamin all three constructs. The first transgenic line (referred to asRBS-HSA) has an operon formed by the aadA gene, followed by the hsagene, whereas the second transgenic line (5′UTR-HSA) harbored anexpression cassette that contained the aadA gene under thetranscriptional regulation of the Prrn promoter, as well as the hsa geneunder the transcriptional regulation of the psbA promoter and thetranslational enhancement of the 5′ psbA UTR. This transgenic line waspredicted to produce a monocistronic hsa transcript (FIG. 47A). Finally,the third transgenic line (ORF-HSA) contained a four-gene operon formedby the aadA, and hsa genes, as well as the orf1 and orf2 sequences ofthe cry2Aa2 operon from the bacterium Bacillus thuringiensis (FIG. 47A).

Northern blot analyses of the RBS-HSA lines with the hsa and the aadAprobes revealed that the most abundant transcript was a dicistron of apredicted 2.8 knt (aadA-hsa, FIG. 47B, D, b), followed by twopolycistronic transcripts: one transcribed from the native 16S Prrnpromoter of an expected size of 5.3 knt (FIG. 47B, D, h), and the3.2-knt transcript transcribed (FIG. 47B, D, d) from the engineered Prrnpromoter, terminating downstream of the 3′UTR of the gene cassette. Nomonocistrons were detected in these RBS-HSA transgenic lines.Quantification of transcripts from the northern blots obtained with thehsa probe revealed that the polycistrons transcribed from the engineeredPrrn promoter accounted for 65.5±3% of the transcript detected in theselines (FIG. 47C). The polycistrons terminating downstream of the 3′UTRin the trnA region and the one transcribed from the native 16S Prrn were24.9±2. and 9.6±1% of the total transcripts, respectively (FIG. 47C, d,h). Values for the northern analysis performed with the aadA probe weresimilar, with 61.8±3% for the polycistron transcribed from theengineered Prrn promoter, 31.7±3% and 6.5±0.3% for the read-throughtranscript and the polycistron transcribed from the native promoter,respectively (FIG. 47E, b, d, h). This analysis shows that there isabundant read-though transcription.

The ORF-HSA transgenic lines also showed a similar transcription patternwith respect to the RBS-HSA line when probed with the hsa or aadA probe.When hybridized with the orf1,2 probe, the same pattern to that obtainedwith the aadA probe was observed, and no processing was detected betweenorf2 and the hsa gene (FIG. 47F, lanes 7-9). The most abundanttranscript was the polycistron containing all four genes (predicted sizeof 4.4 knt), which was transcribed from the engineered Prrn promoter,representing 68.1±2%, 65.5±1% and 43.3±4% of the total transcriptsdetected with the hsa, aadA or orf1,2 probes, respectively (FIG. 47B, D,F, f). Additionally, the predicted 6.9 knt polycistron originating atthe Prrn native promoter was also detected (FIG. 47D, k) and thisrepresented 6.8±0.4% of the polycistrons (FIG. 47E, k). A ˜5.2 knttranscript (FIG. 47B, D, F, g) obtained from the engineered 16S Prrn andprocessed downstream of the 3′UTR was also observed. This transcript wasabout 27.7±1. % and 31.9±2% of the polycistrons detected with the aadAor hsa probes (FIG. 47C, g, and E, g), respectively, and 44.2±1% ofthose detected with the orf1,2 probe (FIG. 47G, g). Finally, transgeniclines engineered with the aadA-5′UTR-hsa construct produced transcriptsabout 200 nt longer than the transgenic lines transformed with theaadA-hsa construct (FIG. 47B, D, c, e). This increase in transcript sizeis due to the presence of the psbA 5′UTR and promoter. Additionally,this transgenic line produced an abundant hsa monocistron (2.1 knt) thataccounted for approximately 50% of the total transcript detected withthe hsa probe (FIG. 47B, C a); this transcript was not detected with theaadA, nor with the orf1,2 probes, (FIG. 47D, lanes 7-9). Thepolycistrons transcribed from the engineered Prrn and native promoterwere 28.7±3% and 9.4±2% of the transcripts produced (FIG. 47E, g, k)Similar transcript abundance was detected in northern blot analyses inwhich the aadA probe was used (FIG. 47E, g, k). Furthermore,read-through transcripts processed downstream of the transgene cassette,in the trnA native gene, were detected (FIG. 47B, D letters e, j). Thecombined abundance of these transcripts was 15.5% (FIG. 47C, e, j),whereas the overall polycistron abundance in this transgenic line was asmuch as the monocistronic transcript.

When RNA from the different transgenic hsa lines were hybridized withthe psbA 3′UTR probe, a pattern similar to that obtained with the hsagene-specific probe was observed (FIG. 51A). Because the native psbAtranscript was also detected, the abundance of both native andheterologous operons could be observed. However, endogenous versusheterologous transcript abundance could only be compared amongtranscripts that were regulated by the same psbA promoter (FIG. 51A,lanes 4-6a). The results showed that the 5′UTR-HSA monocistronictranscript was approximately 1.6 times as abundant as that of the nativepsbA. This may be due to the effect of gene dosage, as the trangene isintegrated into the inverted repeat region, whereas the psbA gene islocated in the large single copy region.

Western blot analyses of the different constructs showed expression ofthe HSA monomer (66 kDa) and dimer (132 kDa) in the transgenic linesharboring the 5′UTR-hsa and the orf1,2-hsa constructs (FIG. 47H).Transgenic lines expressing the monocistrons showed expression levelssimilar to the ORF-HSA transgenic line, in which only polycistrons weretranslated. The abundant translation of ORF2 protein (45 kDa) from theaadA-orf1-orf2-hsa transgenic lines (FIG. 47I), which only transcribedtricistrons and polycistrons, support the view that polycistrons arehighly stable in the chloroplast and can be efficiently translatedwithout further processing. This was also observed in polysomefractionation assays, in which larger polycistronic transcripts of theORF-HSA lines were detected in the lower fractions of the gradient (datanot shown). Expression of the hsa gene in the transgenic line ORF-HSA atlevels similar to the ones produced by the psbA-5′UTR-hsa transgeniclines, suggest a similar translation efficiency for heterologouspolycistrons and monocistrons in the chloroplast (FIG. 47H).

The accumulation of human serum albumin in transgenic linesaadA-orf1-orf2-hsa was monitored under different photoperiods anddevelopmental stages by performing ELISA analyses. These experimentswere conducted to determine whether hsa expression under the cry 5′UTR,which is a heterologous 5′UTR, is light dependent or developmentallyregulated. The data obtained from the analysis of cell extracts fromyoung, mature and old leaves exposed to periods of 0, 4, 8, and 16 hoursof light, revealed no significant differences among age of leaf or amongdifferent periods of illumination. Therefore, HSA accumulation in thistransgenic line regulated by a heterologous 5′ UTR is independent oflight regulation and is free of cellular control (FIG. 48).

Transcription and Translation of the tps1 Operon

The tps1 gene coding for trehalose phosphate synthase was engineeredinto a two-gene operon, formed by the aadA and the tps1 genes, andtranscribed from the engineered Prrn promoter (RBS-TPS1 lines) (FIG.49A). Northern blot analyses with either the tps1-specific oraadA-specific probes detected the expected 2.7 knt dicistron (aadA-tps1)as the most prominent transcript (FIG. 49B, D, a). Densitometricanalyses of the northern blots showed that the aadA-tps1 dicistronaccounted for 43.3±3% of the total transcripts detected by the tps1probe, and 59.8±5% of the total transcript when the aadA probe was used(FIG. 49C, a, and E, a). A predicted 5.2 knt polycistron observed in thenorthern blots with either the tps1 and aadA probes (FIG. 49B, c and D,c), is transcribed from the native 16S Prrn (FIG. 49A, c). Additionally,the tps1 probe detected less abundant polycistrons of about 3.5 knt(FIG. 49B, D, b) and ˜6.5 knt (FIG. 49B, d), transcribed from theengineered Prrn promoter and the native 16S Prrn promoter, respectively,terminating downstream of the 3′UTR. The 3.5-knt polycistron was alsodetected by the aadA probe. Transcripts that ended in the trnA intronregion (downstream of the engineered 3′UTR) were also detected in thecry (FIG. 45B, b and D, e) and hsa transgenic lines (FIG. 47B, d, e, g;D d, e, g, and F, g), indicating that this region may contain differentprocessing sequences. The transcripts processed at the trnA locationaccount for about 37% of the total transcripts detected in thetransgenic lines (FIG. 49C b, d and E, b). Transcripts longer than the6.5 knt polycistrons may terminate at undetermined locations and thesewere not quantified densitometrically. No monocistron was detected inthe northern blots with the tps1 probe nor with the aadA probe,indicating that the polycistron is not being processed in thesetransgenic lines, whereas the larger transcripts detected are likely tobe read-through.

Northern blot analyses performed using the psbA 3′UTR probe (FIG. 51C,lanes 1-3), revealed a pattern consistent to that obtained using thegene-specific tps1 probe (see FIG. 49B). In addition, the native psbAtranscript was also detected and was similar in abundance to theaadA/tps1 dicistron (FIG. 51C, a). However, transcript abundance cannotbe quantitatively compared because they are regulated by differentpromoters. Larger, less abundant transcripts (FIG. 51C, b-c) were alsodetected with the gene-specific tps1 probe (see FIG. 49B), and maycorrespond to read-through transcripts.

Western blot analyses performed to detect the trehalose phosphatesynthase revealed efficient translation of polycistrons, as shown by theabundant accumulation of a 65 kDa polypeptide corresponding to thisprotein (FIG. 49F). Because no monocistrons for tps1 or aadA weredetected in the northern blot analyses, hyperexpression of TPS1 shouldthus be the result of efficient translation of polycistrons intransgenic chloroplasts.

Transcription and Translation of the ctb Operon

RNA from chloroplast transgenic lines transformed with the aadA-ctxb(referred to as RBS-CTB lines) or 5′UTR-ctxb-gfp fusion constructs(5′UTR-CTB-GFP lines) were also analyzed by northern blots. The RBS-CTBlines showed dicistrons and polycistrons, whereas the 5′UTR-CTB-GFPtransgenic lines showed monocistrons along with several polycistrons.Predicted dicistrons of 1.3 knt (FIG. 50B, a and D, a) and 2.3 knt (FIG.50B, d and D, d) transcribed from the engineered Prrn promoter weredetected with either the ctxb or aadA probe. Additionally, polycistronstranscribed from the native 16S Prrn were observed in both transgeniclines. In the RBS-CTB transgenic lines, the aadA-ctxb polycistron was ofa predicted size of 3.8 knt (FIG. 50B, f and D, f), while in the 5′UTR-CTB-GFP transgenic line aadA-5′-UTR-ctxb-gfp polyciston was 4.8 knt(FIG. 50B, g and D, g); both polycistrons code for four genes (16 rRNAgene, trnI gene plus the two heterologous genes). Polycistronictranscripts of higher molecular weight appear to terminate downstreamfrom the engineered 3′UTR (FIG. 50B, D, i, h), as well as thetranscripts of ˜2.2 knt (FIG. 50A,B, c) and ˜3.5 knt (FIG. 50A,B,e),obtained from the engineered Prrn promoter and processed downstream ofthe 3′UTR in the gene construct. The cxtb-gfp monocistron of 1.4 knt(FIG. 50B, b) was detected with the ctxb probe but not with the aadAprobe; besides this transcript, no other monocistron was detected inthese analyses. Its average relative abundance was 42.1±3% of the totalheterologous transcripts in the 5′UTR-CTB-GFP transgenic lines (FIG.50C, b), while the total combined abundance of the polycistrons averaged56% (FIG. 50C, d, g), with the polycistron transcribed from theengineered Prm accounting for 22.9±1% of the total transcripts (FIG.50C, d). For the RBS-CTB transgenic line, 100% of the transcripts werepolycistrons, of which the most abundant transcript was the aadA-ctxBdicistron, (about 45% of the total transcripts), followed byapproximately 30% of the polycistron transcribed from the engineeredPrrn and processed downstream at the trnA gene (FIG. 50D, a. c and E, a,c).

Additional northern blot analyses performed with the psbA 3′UTR probe(FIG. 51D) revealed a transcript pattern consistent to that obtainedwith the ctxB gene-specific probe (see FIG. 50B). Furthermore, thenative psbA transcript was also detected. Because the size of theaadA/ctxb dicistron (1.25 knt) is similar to that of the endogenous psbA(1.3 knt), they could not be distinguished from each other. However,this may account for the increase in transcript abundance observed inrelation to the native psbA transcript (FIG. 51D, lanes wt and 1-3a*).Due to similar reasons, the increase in transcript abundance of thenative psbA transcript observed on the 5′UTR-CTB-GFP transgenic lines(FIG. 51D, lanes 4-6b*) could be due to the presence of the ctxb-gfpmonocistron (1.4 knt). Although, the native psbA and the ctxb-gfp genesare regulated by the same psbA promoter, transcript abundance could bequantitatively compared. However, because both transcripts are similarin size, comparison between these native and heterologous transcriptswas not possible.

The western blot analyses showed that transgenic lines expressing eitherCTB or CTB-GFP fusion produced large amounts of either protein (FIG.50F, G). CTB protein was detected as a higher molecular weightpolypeptide (trimer of 35 kDa) than the E. coli expressed CTB (FIG.50F). High protein level was also detected for the gfp-ctxB fusionprotein (FIG. 50G), which was detected in the monomeric form (45 kDa).Interestingly, expression levels in both transgenic lines were similar,even though in the aadA-5′UTR-ctxb-gfp transgenic line the more abundanttranscript was the monocistron. This again suggests that polycistronsare translated as effectively as monocistrons.

Discussion

The chloroplast genome has been engineered with single genes to conferuseful agronomic traits including herbicide resistance (Daniell et al.,1998), insect resistance (McBride et al., 1995; Kota et al., 1999, DeCosa et al., 2001), disease resistance (DeGray et al., 2001), droughttolerance (Lee et al., 2003), salt tolerance (Kumar et al., 2004a), andphytoremediation (Ruiz et al., 2003). Recent success in transforming thechloroplast genome of several major crops, including cotton (Kumar etal., 2004b) and soybean (Dufourmantel et al., 2004) has opened thisfield for commercial development. Because most of the desired traitsrequire multigene engineering, it is important to understandtranscription, posttranscriptional changes and translation ofheterologous polycistrons within plastids.

Transcript analyses performed in this study repeatedly confirmed thatdifferent transgenic lines harboring multigenic operons generatedpolycistrons as the most abundant transcript form, along withmonocistronic mRNA. This observation is further supported by thepolysome fractionation assays performed on the cry2Aa2 samples, in whichlarger transcripts were collected from the fractions associated topolyribosomes. Smaller transcripts were observed mainly in the upperfractions of the gradient, suggesting that polycistrons may bepreferentially translated without processing. Similar results wereobtained after stripping the membranes and re-probing with the orf1,2probe. Polycistronic polysomal RNA has been previously reported innative chloroplast operons, as well as multiple open reading framessimultaneously translated from polycistions (Barkan, 1988), however, insuch case, polycistronic transcripts were less abundant thanmonocistrons. In the case of the cry2Aa2 operon, ribosome-associatedpolycistrons were in much higher abundance than the monocistronictranscripts, suggesting that the heterologous operon is preferentiallytranslated as a polycistronic unit. Similar results were observed withchloroplast transgenic lines harboring the aadA-orf1-orf2-hsa operon(data not shown). These observations contrast with the general consensusfor native chloroplast translation mechanisms (Barkan, 1988; Barkan etal., 1994; Zerges, 2000; Meierhoff et al., 2003), thus showing thatmultigene operons engineered into the chloroplast genome do notnecessarily require processing of polycistrons to monocistrons ordicistrons for efficient translation.

Processing was observed in the native cry2Aa2 operon, between orf2 andthe cry2Aa2 genes on the transgenic lines. However, this event did notoccur between orf1 and orf2 of this operon, or at intergenic sequencesof the other engineered operons studied. The fact that processingoccurred only in the cry2Aa2 5′UTR suggests that this intergenicsequence might contain unique information required for processing. Byusing computer simulation, it was observed that the heterologousbacterial intergenic transcript sequences, may form secondarystructures. Evidence for the protection of chloroplast RNA by 5′UTRs hasbeen previously discussed (Drager et al., 1998), as well as the role of5′UTR secondary structures in RNA stability (Zou et al., 2003).Additionally, previous reports have shown that, intergenic sequencesforming stable secondary structures that mask the ribosome-binding site,may affect the translation of the downstream gene (Barkan et al., 1994;Hirose and Sugiura 1997; Del Campo et al., 2002). These observationsoffer the possibility of further studies involving the role ofintergenic secondary structures of native and heterologous operons inpost-transcriptional processes.

Transgenic lines harboring the engineered aadA-orf1-orf2-hsa operonshowed no difference in HSA accumulation in response to light or darkconditions, in contrast to those transformed with the hsa gene andnative psbA 5′ UTR. This suggests that the translation enhancementobserved is not light-dependent. Thus, the heterologous cry2aA2 operonUTR region is independent of nuclear and chloroplast control, unlike thepsbA regulatory sequences (Fernandez San Millan, et al., 2003; Zerges,2004). Such heterologous UTRs have played a major role in transgeneexpression in non-green tissues, such as carrot roots (Kumar et al.,2004a), or in non-green cultured cells (Kumar et al., 2004 a, b), tofacilitate transformation of recalcitrant crops.

Data shown here supports the idea that engineered operons in thechloroplast, which do not carry any intergenic sequences capable offorming stable secondary structures, can be translated very efficientlyand do not require processing into monocistrons in order to betranslated. The processing observed in the cry2Aa2 transgenic lines maybe due to endonucleolytic cleavage of a region in the intergenicsequence, but it does not indicate that this processing has to occur inorder for translation to take place. An interesting observation is thatthe aadA-orf1-orf2 tricistron produced by the processing event does notcontain a 3′ UTR region, yet this transcript is as abundant as thepolycistrons, which contain the 3′UTR and are efficiently translated.This shows that polycistrons may be stable in the chloroplast, even inthe absence of the 3′UTR.

In chloroplasts, all of the genes in the 16S rrn operon, including thetrnA, trnI, as well as 23S, 4.5S and 5S rrn genes (which are downstreamof the integrated transgenes), are transcribed from the native Prrnpromoter. Therefore, disruption of these polycistrons by the insertionof the foreign operon due to effective termination at the 3′untranslated regions would mean that the trnA and other downstream geneswould not be transcribed, affecting chloroplast protein synthesis.However, this was not the case; all the transgenic lines grew similar tothe wild type plants, indicating that the read-through transcriptsformed by the insertion of foreign operons were sufficient for optimalribosome synthesis in chloroplasts. Read-through transcripts processedat the trnA region accounted for about 26 to 39% of the totalheterologous transcripts in all transgenic lines tested whereas, inHSA-expressing transgenic lines, this percentage was between 15% and32%. Introns within the trnA gene undergo splicing and otherposttranscriptional modifications in order to produce the functionaltrnA (Barkan et al., 2004). Therefore, such processing may modifypolycistronic transcripts that read through from the 3′UTR psbAengineered in these chloroplast vectors. Additionally, largerpolycistrons were also detected, although these were not quantified.

The transcript profile for the transgenic lines 5′UTR-hsa and5′UTR-ctxb-gfp, (the only two transgenic lines in this study thattranscribed monocistrons) was very similar The monocistronic transcriptsaccounted for about 42% to 50% of the total heterologous transcriptsexamined. The total polycistronic levels in these two transgenic lines,including read-through transcripts were between 50% and 57%. In all thetransgenic lines that did not transcribe monocistrons, the most abundanttranscript was transcribed from the engineered Prm promoter, terminatingat the 3′UTR, which accounted for 43% to 59% of the total transcriptsdetected.

Data generated by analyzing the different transcripts with the psbA3′UTR probe not only supported the previous results observed with thegene-specific probes, but also allowed comparison with the native psbAtranscripts. In two transgenic lines (5′UTR-HSA and 5′UTR-CTB-GFP), thepsbA 5′UTR was used upstream of the genes of interest. In such cases,endogenous versus heterologous transcript abundance could bequantitatively compared, unless the 5′utr-ctxb-gfp transcript wassimilar in size to the native psbA gene. Comparison of the native psbAand 5′utr-hsa transcripts showed a greater abundance (1.6 times) of theheterologous transcript. This could be attributed to gene dosage, as theheterologous operons are integrated into the inverted repeat region,whereas the native psbA gene is located in the single-copy region. Inaddition, transcript abundance was variable among the remainingheterologous operons regulated by the 16S prrn promoter. Variabilitycould be attributed to differences in mRNA stability, as well as in thelevel of posttranscriptional processing of the primary transcripts(Barkan and Goldschmidt-Clermont, 2000; Monde et al., 2000b; del Campoet al., 2002).

The ability to engineer foreign genes without promoters or otherregulatory sequences has several advantages. Also, repeated sequencesmay cause deletion of the transgene (Iamtham and Day, 2000).Observations reported here show evidence for transcription andprocessing of heterologous operons. While endogenous polycistronsrequire processing for effective translation, this is not required forexpression of foreign operons. Native polycistrons require chloroplastspecific 3′UTRs for stability, which is not always required forheterologous polycistrons. Untranslated regions in native transcriptsare regulated by nuclear factors, whereas heterologous transcripts havenot been shown to be dependent of such regulations. Specificnuclear-encoded factors recognize sequences in native transcripts forthe processing of primary mRNA (Barkan, 2004). This is not the case inforeign operons where heterologous sequences can be recognized andprocessed by the chloroplast posttranscriptional machinery. Finally, inboth native and foreign operons there are abundant read-throughtranscripts that allow the expression of genes downstream of 3′UTRs.Addressing questions of the translation of polycistrons and sequencesrequired for transcript processing and stability is essential forchloroplast metabolic engineering. Knowledge of such factors wouldenable engineering pathways that will not be under the complexpost-transcriptional regulatory machinery of the chloroplast.

One of the primary advantages of using heterologous sequences forincreasing gene expression is the lack of cellular control over thesesequences, allowing the enhancement of transgene expression in green andnon-green tissues. Recently, the use of the g10 5′UTR facilitated thetransformation of non-green plastids of carrot (Kumar et al., 2004a).Additionally, the use of a gene cassette containing the selectablemarker genes under the regulation of heterologous UTRs, increasedtransformation efficiency and facilitated cotton plastid transformation(Kumar et al., 2004b). Recent accomplishments in the transformation ofagronomically important species through somatic embryogenesis usingspecies-specific chloroplasts vectors, also broadens the possibility ofextending this technology to crops that have been, until now,recalcitrant to chloroplast transformation (Dufourmantel et al., 2004;Kumar et al., 2004a; Kumar et al., 2004b; Daniell et al 2005).

In this study, we report the translation of polycistronic transcriptswithout processing, the expression of multigene operons independently ofcellular control, and the stability of heterologous polycistrons lackinga 3′UTR. These results suggest that it is possible to effectivelyexpress multiple genes via the chloroplast genome without significantintervention of chloroplast regulation. The findings of this studyfacilitate multigene engineering via the plastid genome in both greenand non-green plastids. One embodiment of the invention relates to avector suitable for integration into the chloroplast genome thatcomprises multigene operons, a plant stably transformed with such avector and a method of transforming a plant with such a vector.

The results reported here are the first attempts to understand multigeneengineering in transgenic plastids.

Materials and Methods Chloroplast Transformation, Selection andCharacterization of Transgenic Plants

The chloroplast transformation, selection and characterization of thetransgenic lines used in this study have been previously reported(Daniell et al., 2001; De Cosa et al., 2001; Lee et al., 2003;Fernandez-San Millan et al., 2003) with the exception of the ctb-gfptransgenic lines. Sterile tobacco leaves were bombarded using theBio-Rad PDS-1000/He biolistic device as described previously (Daniell,1997; Daniell et al., 2004a, b).

Chloroplast Expression Vector Carrying the hsa Gene.

The pLDA-sdHSA vector was constructed by inserting the hsa gene (1.8 kb)into EcoRI/NotI sites of the multiple cloning site of the chloroplasttransformation vector (pLD-ctv). This construct contained the hsa geneand a ribosome binding site sequence (ggagg) upstream of the gene. Forthe pLDA-5′UTR-hsa vector, the promoter and 5′UTR (205 bp) from psbAgene were amplified by PCR from tobacco chloroplast DNA and thensequenced. The subsequent in-frame cloning of the promoter/5′UTRupstream and hsa gene into pLD-ctv vector by EcoRI/NotI digestionproduced the functional gene cassette.

Chloroplast Expression Vector Carrying the ctxB Gene.

A ribosome binding site (GGAGG) was engineered five bases upstream ofthe start codon of the ctxB gene. The PCR product was then cloned intopCR2.1 vector (Invitrogen) and subsequently cloned into the chloroplasttransformation vector (pLD-ctv) after the sequencing of the open readingframe. The pLD vector carrying the ctxB gene was used for successivetransformation of tobacco chloroplast genome according to the publishedprotocol.

Chloroplast Expression Vector Carrying the tps1 Gene.

The yeast trehalose phosphate synthase (tps1) gene was inserted into theXbaI site of the universal chloroplast expression (pCt) vector betweenthe aadA selection marker gene for spectinomycin resistance and the psbAterminator to form the final pCt-tps1 vector.

Chloroplast Expression Vector Carrying the Cry2Aa2 Operon.

The cry2Aa2 operon from the HD-1 strain (Delattre et al., 1999) wasinserted into the universal chloroplast expression vector, pLD ctv, toform the final shuttle vector pLD-BD Cry2Aa2 operon (De Cosa et al.,2001). This vector contains the 16S ribosomal RNA (rRNA) promoter (Prrn)upstream of the aadA gene (aminoglycoside 3′-adenylyltransferase) forspectinomycin resistance, the three genes of the cry2Aa2 operon, and thepsbA terminator from the 3′ region of the chloroplast photosystem IIgene.

Plant Transformation

Tobacco leaves were transformed by particle bombardment (Bio-RadPDS-1000He device), using 0.6 nm gold microcarriers coated with thepCt-TPS1 chloroplast expression vector, and delivered at 1,100 psi witha target distance of 9 cm (Daniell, 1997). The bombarded leaves wereselected on RMOP medium containing 500 ng/ml spectinomycin to regeneratethe transformants, as previously described (Kumar and Daniell, 2004;Daniell et al., 2004a)

Northern-Blot Analysis

Total plant RNA from untransformed tobacco (var. Petit Havana) and fromthree clones of T₁ chloroplast transgenic tobacco plants, was isolatedby using the RNeasy Mini Kit (Qiagen, Valencia, Calif.) and protocol.Northern blot analyses were performed essentially as follows. Total RNA(1 μg) per plant sample was resolved in a 1.2% (w/v)agarose/formaldehyde gel at 55 V for 2.5 h. The RNA was transferredovernight to a nitrocellulose membrane by capillarity. The next day, themembrane was rinsed twice in 2×SSC (0.3 M NaCl and 0.03 M sodiumcitrate), dried on Whatman paper, and then cross-linked in the GS GeneLinker (Bio-Rad, Hercules, Calif.) at setting C3 (150 njouls).

The probes used for northern blot analyses were obtained as follows: theaadA (aminoglycoside 3′ adenylyl transferase) probe was obtained byBstEII/XbaI restriction digestion of plasmid pUC19-16S/aadA; the ctxB(cholera toxin β-subunit) and tps1 (trehalose phosphate synthase) probeswere obtained by XbaI restriction digestion of plasmids pSBL-CTB andpSBL-TPS1, respectively. The Cry2Aa2 (Bacillus thuringiensisinsecticidal protein) probe was obtained by XbaI digestion of plasmidpSBL-ctv-CryIIA. The hsa (human serum albumin) probe was obtained byEcoRV/NotI digestion of plasmid pCR2.1 ATG-HSA. Finally, the orf 1,2probe was obtained by EcoRI digestion of plasmid pCR2.1 ORF1,2 and thepsbA 3′UTR probe was obtained by pstI/XbaI digestion of plasmid pLD-ctv.

Probes were radio labeled with ³²P dCTP by using Ready Mix® and QuantG-50® micro columns for purification (Amersham, Arlington Heights,Ill.). Prehybridization and hybridization were performed using theQuick-Hyb® solution (Stratagene, La Jolla, Calif.). The membrane wasthen washed twice for 15 min at room temperature in 2×SSC with 0.1%(w/v) SDS, followed by two additional washes at 60° C. (to increase thestringency) for 15 min with 0.1×SSC with 0.1% (w/v) SDS. Radiolabeledblots were exposed to x-ray films and then developed in the Mini-MedicalSeries x-ray film processor (AFP Imaging, Elmsford, N.Y.). Whenrequired, membranes were stripped by applying boiling 0.1% SSC and 0.1%SDS to the membrane, washing for 15 minutes, and repeating beforere-hybridizing with a different probe.

Relative transcript levels within each lane were measured by spotdensitometry (Alphaimager 3300, Alpha Innotech, San Leandro, Calif.) onradiograms from the different northern blot analyses, except thoseobtained using the psbA 3′UTR probe. The former are shown as percentageof abundance within each line and therefore comparison among linescannot be made. For the blots obtained by hybridization with the psbA3′UTR probe, transcript abundance was quantified by using the wild-typenative psbA transcript, to which a value of 1 was assigned. All othertranscripts show values greater or smaller than 1, depending onabundance in relation to the wild-type psbA transcript, and are shown asadditive values in each line. Average transcript abundance wascalculated among corresponding clones and the standard deviation wasdetermined

Polysomal Fractionation Assays

Approximately 0.3 grams of leaf material from transgenic tobaccoharboring the cry2Aa2 operon were thoroughly ground in liquid nitrogenand resuspended in polysome extraction buffer (Barkan, personalcommunication). The ground tissue was treated according to the protocoldescribed by Barkan (1988), with some modifications. The samples weretreated with 0.5% sodium deoxycholate and loaded onto 15%-55% sucrosegradients and centrifuged at 45,000 rpm for 65 minutes (Beckmann rotorSW52Ti). Fractions were collected from the bottom of the tube ontomicrocentrifuge tubes containing 50 μl 5% SDS and 0.2M EDTA, up to avolume of about 500 μl for each fraction. Polysomal RNA was extractedwith phenol:chloroform:isoamyl-alcohol (25:24:1), followed by ethanolprecipitation. The resulting pellets were resuspended in RNase-free TEbuffer (pH 8.0) and stored at −80° C., or loaded onto denaturing 1.2%agarose-formaldehyde gels (5 μl from each fraction). Northern blotanalyses were then performed as described above. Blots were hybridizedwith cry2Aa2 and orf1,2 probes.

To provide further evidence that the RNA obtained from the bottomfractions of the sucrose gradient corresponded to polysome-associatedRNA, a puromycin-release control was included. Before treatment withsodium deoxycholate, samples were treated with 150 μl of 2M KCl and 170μl of puromycin (3 mg/ml stock) and incubated 10 minutes at 37° C.Sodium deoxycholate was then added to 0.5%, and the samples wereincubated 5 minutes on ice before loading onto the sucrose gradient. RNAextraction and Northern blot analyses were performed as described above.The blots were hybridized with the aadA and orf1,2 probes.

Western-Blot Analyses

Protein samples were obtained from 100 mg of leaf material from the samewild type and transgenic lines used in the Northern analyses by grindingthe tissue to a fine powder in liquid nitrogen. Subsequenthomogenization in 200 μl plant protein extraction buffer (100 mM NaCl,10 mM EDTA, 200 mM Tris-HCl, 0.05% (w/v) Tween-20, 0.1% (w/v) SDS, 14 mMβ-mercaptoethanol (BME), 400 mM sucrose and 2 mM phenylmethylsulfonylfluoride) was performed, followed by a centrifugation step at 15.7×g for1 minute to remove solids. The Bacillus thuringiensis Cry2Aa2 proteinwas extracted from 100 mg of transgenic leaf material by adding 200 μlof 50 mM NaOH to solubilize the Cry protein from the crystals formed inthe transgenic plants, and centrifuged at 10,000×g for 1 minute toremove cell debris. Total protein concentrations for the samples weredetermined by Bradford assay (Bio-Rad Protein Assay) with bovine serumalbumin as the protein standard.

Approximately 60 μg of total soluble protein was loaded onto 12% v/vSDS-polyacrylamide gels and separated by electrophoresis. The separatedproteins were then transferred to a nitrocellulose membrane (Bio-Rad,Hercules, Calif.). The membrane was blocked for 1 hr with PTM buffer:1×PBS (phosphate buffer solution), 0.05% (v/v) Tween-20 and 3% (w/v)non-fat dry milk The membranes were probed with primary and secondaryantibodies as follows: for 2 hrs with primary antibody, then rinsed withwater twice and probed with secondary antibody for 1.5 hrs. Finally, themembranes were washed 3 times for 15 minutes with PT buffer (1×PBS,0.05% (v/v) Tween-20) and one time with 1×PBS for 10 minutes, followedby incubation in Lumi-phos® WB (Pierce, Rockford, Ill.) reagent for thealkaline phosphatase reaction or SuperSignal (Pierce) reagent forhorseradish peroxidase (HRP) reaction.

Film exposure took place for 1, 3, 5 or 10 minutes, depending on thestrength of the signal of each blot. The antibodies used and theirrespective dilutions were the following: anti-cry2A (Envirologix,Portland, Me.), dilution 1:3,000; anti-ORF2 (Moar et al., 1989),dilution 1:1,000; anti-HSA (Sigma, St. Louis, Mo.), dilution 1:3,000;anti-CTB (Sigma), dilution 1:2,500; anti-PA (Dr. Stephan Leppla, NIH),dilution 1:30,000. Secondary antibodies were used as follows: alkalinephosphatase conjugated anti-rabbit antibody (Sigma) was used to probeagainst every primary antibody with the exception of anti-PA which wasprobed with HRP conjugated anti-mouse antibody; dilutions of 1:5,000anti-rabbit antibody were used for anti-HSA, anti-ORF2 and anti-Cry2A,for anti-CTB the dilution was 1:4,000. Anti-mouse antibody was used in a1:5,000 dilution.

ELISA Quantification

The Human Albumin Quantitation Kit (Bethyl Laboratories) was used forELISA quantification. Leaf material (100 mg) of the aadA-orf1-orf2-hsatransgenic line was ground in liquid nitrogen and resuspended in 700 μlof 50 mM NaOH. The leaf extracts were then diluted to fit in the linearrange of the provided HSA standard. Absorbance was read at 450 nm. TheDC protein assay (Bio-Rad) was used to determine total soluble proteinconcentration following the manufacturer's protocol.

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Example 7 Efficacy and Functionality of Chloroplast-Derived AnthraxProtective Antigen Introduction

Anthrax, a fatal bacterial infection, is caused by Bacillus anthracis, agram-positive spore-forming organism. It is a zoonotic diseasetransmitted from animals to humans. CDC lists Bacillus anthracis as acategory A biological agent due to its severity of impact on humanhealth, high mortality rate, acuteness of disease, and potential fordelivery as a biological weapon. The disease is acquired when sporesenter the body through the skin or by inhalation or ingestion. Virulentstrains of B. anthracis contain plasmids pX01, which carries genesencoding the toxins, and pX02, which encodes the poly-Dglutamic acidcapsule. Plasmid pX01 carries the genes pagA, lef, and cya that encodethe protective antigen (PA), lethal factor (LF), and edema factor (EF),respectively. The term “protective antigen” is derived because of thisprotein's ability to elicit a protective immune response againstanthrax. None of these proteins is toxic when administered individuallyto cells or animals. However, PA in combination with EF, known as edematoxin, causes edema. Similarly, PA in combination with LF forms lethaltoxin (LT) (1, 5).

PA is the primary immunogen and key component of human vaccines producedand licensed in the United Kingdom and United States. The current U.S.vaccine (BioThrax; BioPort Corp.) consists of an alum-absorbed,formalin-treated culture supernatant of a toxigenic, nonencapsulatedstrain of B. anthracis. The British anthrax vaccine is produced fromsupernatant of a static culture of the Sterne strain, a nonencapsulatedtoxigenic variant of B. anthracis, adsorbed to aluminum salts. Thesevaccines contain predominantly PA, but also small quantities of LF andtrace amounts of EF (31). These traces of LF and EF may contribute tothe vaccine side effects, such as local pain and edema (19), andrelatively high rates of local and systemic reactions, includinginflammation, flu-like symptoms, malaise, rash, arthralgia, and headache(14, 29). Therefore, an effective expression system that can provide aclean, safe, and efficacious vaccine is required. Recombinant PA hasbeen expressed in Escherichia coli (15), Lactobacillus casei (32), andSalmonella enterica serovar Typhimurium (6). Expression of PA in plantsthrough chloroplast transformation has several advantages over bacterialand mammalian expression systems. Foreign proteins have been expressedat extraordinarily high levels in transgenic chloroplasts due to thepresence of 10,000 copies of the chloroplast genomes per cell. Theseinclude A T-rich proteins such as Cry2a (67% AT) at 47% of the totalsoluble protein (TSP) (11), cholera toxin B chain fusion protein (59%AT) at 33% TSP (23), and human serum albumin (66% AT) up to 11.1% TSP(12). Therefore, we first tested the feasibility of expressing PA intransgenic chloroplasts (30), but no further studies were possiblebecause no tag was used in that study to facilitate purification. Inaddition to high levels of transgene expression, there are several otheradvantages to chloroplast genetic engineering. Several genes can beintroduced in a single trans-formation event to facilitate developmentof multivalent vaccines (11, 28). Gene silencing is a common concern innuclear transformation, but this has not been observed in transgenicchloroplasts in spite of hyperexpression of transgenes (11).

There is minimal risk of animal or human pathogens contaminating thevaccine as seen with mammalian expression systems. Additionally,chloroplast expression systems minimize cross-pollination of thetransgene due to the maternal inheritance of the chloroplast genome (8).In this study, we expressed PA with a histidine tag in transgenicchloroplasts, characterized the resultant transgenic plants, andperformed immunization studies. We compared the efficacy of theplant-derived PA with that of PA derived from B. anthracis in both invitro and in vivo studies.

Materials and Methods

Construction of pLD-VK1 Vector for Chloroplast Transformation.

The six histidine tag and the factor Xa cleavage site with NdeI and XhoIrestriction sites were introduced N terminal to pagA using PCR (FIG. 52a). The PCR-amplified region was sequenced and shown to matchcorresponding pagA database sequences (accession no. AY700758). The PCRproduct was then cloned into pCR2.1 vector containing the psbA 5′untranslated region (UTR). Finally, the fragment containing the 5′ UTR,His tag, and pagA was cloned into tobacco universal vector pLD-ctv toproduce pLD-VK1 (FIG. 52 a).

Leaf bombardment and selection protocol. Microprojectiles coated withplasmid DNA (pLD-VK1) were bombarded into Nicotiana tabacum var. petitHavana leaves using the biolistic device PDS1000/He (Bio-Rad) asdescribed elsewhere (9). Following incubation at 24° C. in the dark for2 days, the leaves were cut into small (˜5 mm by 5 mm) pieces and placedabaxial side up (five pieces/plate) on selection medium (RMOP[regeneration medium of plants] containing 500 mg/liter spectinomycindihydrochloride [9]). Spectinomycin-resistant shoots obtained afterabout 6 weeks were cut into small pieces (˜2 mm by 2 mm) and placed onplates containing the same selection medium.

Confirmation of Transgene Integration into the Chloroplast Genome.

To confirm the transgene cassette integration into the chloroplastgenome, PCR was performed using the primer pairs 3P(5′-AAAACCCGTCCTCEGTTCGGATTGC-3′) and 3 M(5′-CCGCGTTGTTTCATCAAG-CCTTACG-3′) (10), and to confirm the integrationof gene of interest, PCR was performed using primer pairs 5P(5′-CTGTAGAAGTC-ACCATTGTTGTGC-3′) and 2 M (5′-TGACTGCCCACCTGA-GAGCGGACA-3′) (10).

Southern Blot Analysis.

Two micrograms of plant DNA per sample (isolated using DNeasy kit)digested with BglII was separated on a 0.7% (wt/vol) agarose gel andtransferred to a nylon membrane. The chloroplast vector DNA digestedwith BglII and BamHI generated a 0.8-kb probe homologous to the flankingsequences. Hybridization was performed using the Ready-To-Go protocol(Pharmacia).

Immunoblot Analysis.

Transformed and untransformed leaves (100 mg) were ground in liquidnitrogen and resuspended in 500 μl of extraction buffer (200 mMTris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 2 mMphenylmethylsulfonylfluoride). Leaf crude extracts, boiled (4 min) orunboiled, in sample buffer (Bio-Rad) were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thirty percentacrylamide Bis solution (Bio-Rad) was used to make the 10% gels. The gelwas run in 1× electrode buffer (10× electrode buffer is 30.3 g Trisbase, 144.0 g glycine, and 10.0 g SDS added to 1,000 ml distilledwater). The separated proteins were then transferred to nitrocellulose,and Western blot analyses was performed using anti-PA primary antibody(Immunochemical labs) diluted in phosphate-buffered saline (PBS)-0.1%Tween-3% milk powder (PTM) (1:20,000) and secondary horseradishperoxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG)(Sigma) diluted in PTM (1:5,000) followed by washing with PBS andfinally incubated with Lumiphos WB (Pierce) as a substrate for HRP atroom temperature for 5 min for chemiluminescence.

ELISA for PA.

Leaf samples (100 mg of young, mature, or old leaves) were collectedfrom plants exposed to regular (16 h of light and 8 h of dark) orcontinuous illumination. The extraction buffer (15 mM Na2CO3, 35 mMNaHCO3, 3 mM NaN3, pH 9.6, 0.1% Tween, 5 mM phenylmethylsulfonylfluoride) was used to isolate plant protein. All dilutions were made inthe coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6).Antibodies were used at dilutions similar to those in the Westernblotting protocol. Wells were then loaded with 100 μl of3,3,5,5-tetramethylbenzidine (TMB; American Qualex) substrate andincubated for 10 to 15 min at room temperature. The reaction wasterminated by adding 50 μl of 2 N H2SO4 per well, and the plate was readon a plate reader (Dynex Technologies) at 450 nm.

Purification of His-Tagged PA by Affinity Chromatography.

His affinity chromatography using nickel-chelate-charged columns(Amersham Biosciences) was used to purify His-tagged PA as per themanufacturer's protocol. The buffers used for purification include thefollowing: binding buffer, 20 mM Na2HPO4, 0.5 M NaCl, 10 mM imidazole,pH 7.4; elution buffer, 20 mM Na2HPO4, 0.5 M NaCl, 0.5 M imidazole, pH7.4; and Ni-loading eluent, 100 mM NiSO4 solution (Sigma). Proteinsamples were analyzed for PA using enzyme-linked immunosorbent assay(ELISA). Eluate fractions containing purified PA were pooled togetherand dialyzed against PBS, pH 7.4, using dialysis cassettes (molecularweight, 10,000; Pierce) and concentrated using Centricon10,000-molecularweight-cutoff ultrafiltration units (Millipore)following the manufacturer's protocols.

Macrophage Lysis Assay.

Macrophage lysis assays were performed on the crude leaf extracts,partially purified chloroplast-derived PA, and B. anthracis-derived PA.RAW264.7 macrophage cells were plated in 96-well plates in 120 μlDulbecco's modified Eagle's medium and grown to 50% confluence. Theplant samples or solutions containing 20 μg/ml of the purified PAproteins were diluted serially 3.14-fold in a separate 96-well plate andthen transferred onto the RAW264.7 cells in such a way that the top rowhad plant extract at a 1:50 dilution and PA at 0.4 μg/ml. Cells wereincubated with LT for 2.5 h, and the cell viability was assessed byaddition of MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazoliumbromide] (Sigma, St. Louis, Mo.) at a final concentration of 0.5 mg/ml.Cells were then further incubated with MTT for 40 min, and the bluepigment produced by viable cells was dissolved by aspirating the mediumand adding 50 μl/well of a mixture containing 0.5% (wt/vol) SDS and 25mM HCl in 90% (vol/vol) isopropanol and shaking the plates for 5 minprior to reading at 570 nm using a microplate reader. Control platesreceived medium with no LF to test toxicity of plant material andbuffers.

Immunization Studies in Mice.

The immunization studies were conducted in accordance with federal andinstitutional guidelines. Seven groups of five female 6- to 7-week-oldBALB/c mice (Charles River) were immunized subcutaneously (s.c.; 5 μgPA) at two sites (100 μl per site) on day 0. The groups include miceimmunized with (i) chloroplast-derived PA (CpPA) with adjuvant, (ii)chloroplast-derived PA (CpPA) alone, (iii) Std.PA derived from B.anthracis with adjuvant, (iv) Std-PA alone (26), (v) PA plant leaf crudeextract with adjuvant, or (vi) wild-type plant leaf crude extract withadjuvant and (vii) unimmunized mice. The measurement of PA adsorbed toalhydrogel was done as described previously (20). Booster doses wereadministered on day 14, day 28, and day 140. Blood was drawn from theretro-orbital plexus 15 days after the third and fourth doses (i.e., ondays 43 and 155 of post-initial immunization). The blood samples wereallowed to stay undisturbed for 2 h at room temperature, stored at 4° C.overnight, and centrifuged at 3,000 rpm for 10 min to extract the serum.

ELISA to Detect the Anti-PA IgG Antibodies in the Serum Samples.

Ninety-six well microtiter ELISA plates were coated with 100 μl/well ofPA standard at a concentration of 2.0 _g/ml in PBS, pH 7.4. The plateswere stored overnight at 4° C. The serum samples from the mouse wereserially diluted (1:100 to 1:640,000). Plates were incubated with 100 μlof diluted serum samples for 1 h at 37° C. followed by washing withPBS-Tween. The plates were then incubated for 1 h at 37° C. with 100 μlof HRP-conjugated goat anti-mouse IgG (1:5,000 dilution of 1-mg/mlstock). TMB was used as the substrate, and the reaction was stopped byadding 50 μl of 2 M sulfuric acid. The plates were read on a platereader (Dynex Technologies) at 450 nm. Titer values were calculatedusing a cutoff value equal to an absorbance difference of 0.5 betweenimmunized and unimmunized mice (25).

Toxin Neutralization Assays.

Sera from immunized mice were tested for neutralization in themacrophage cytotoxicity assay described above. LT (PA plus LF) was addedat 50 ng/ml in Dulbecco's modified Eagle's medium to 96-well plates (100μl/well, except 150 μl in first well). Serum from each mouse was diluteddirectly into the LT plates starting at 1:150 and proceeding in3.14-fold dilutions. Each serum was tested in triplicate. Following a30-min incubation of sera with toxin, 90 μl of the mixture was moved toa 96-well plate containing RAW264.7 cells grown to 90% confluence andincubated for 5 h at 37° C. MTT was then added (final concentration, 0.5mg/ml), and cell death was assessed as described above. Neutralizationcurves were plotted, and 50% effective concentrations (EC50s) werecalculated for the averaged data from each mouse serum using GraphPadPrism 4.0 software.

Toxin Challenge in Mice.

Groups of five mice with various immunization treatments described abovewere injected intraperitoneally with 150 μg LT (150 μg LF plus 150 μgPA) in sterile PBS (1 ml). Mice were monitored every 8 h for signs ofmalaise and mortality.

Results and Discussion Chloroplast Vector Design.

The pLD-VK1 vector (FIG. 52 a) contains homologous sequences thatfacilitate recombination of the pag gene cassette between the trnI andtrnA genes of the native chloroplast genome (9). The constitutive 16SrRNA promoter regulates expression of the aadA (aminoglycoside 3′adenyltransferase) gene. The pagA gene is regulated by the psbA promoterand 5′ and 3′ UTRs. The psbA 5′UTR has several sequences for ribosomalbinding that act as a scaffold for the light-regulated proteins involvedin ribosomal binding to enhance translation (12), and the psbA 3′UTRserves to stabilize the transcript.

Demonstration of Transgene Integration.

Several shoots appeared 5 to 6 weeks after the bombardment of tobaccoleaves with gold particles coated with the pLD-VK1 plasmid DNA (FIG. 52a). There are three genetic events that can lead to survival of shootson the selective medium: chloroplast integration, nuclear integration,or spontaneous mutation of the 16S rRNA gene to confer resistance tospectinomycin in the ribosome. True chloroplast transformants weredistinguished from nuclear transformants and spontaneous spectinomycinresistance mutants by PCR. Previously described primers, 3P and 3M, wereused to test for chloroplast integration of transgenes (9). The 3Pprimer anneals to the native chloroplast genome within the 16S rRNAgene. The 3M primer anneals to the aadA gene (FIG. 52 a). Nucleartransformants could be distinguished because 3P will not anneal andmutants were identified because 3M will not anneal. Thus, the 3P and 3Mprimers will only yield a product (1.65 kb) from true chloroplastintegrants (FIG. 52 c).

The integration of the transgenes was further tested by using the 5P and2M primer pairs for PCR analysis. The 5P and 2M primers anneal to theinternal region of the aadA gene and the internal region of the trnAgene, respectively, as shown in FIG. 52 a (9). The product size of apositive clone is 3.9 kb for PA, while the mutants and the control donot show any product. FIG. 52 d shows the result of the 5P/2M PCRanalysis. After PCR analysis using both primer pairs, the transgenicplants were subsequently transferred through different rounds ofselection to obtain mature plants and reach homoplasmy.

Southern Blot Analysis of Transgenic Plants.

The plants that tested positive by PCR analysis were moved through threerounds of selection and were then evaluated by Southern analysis. Theflanking sequence probe (0.81 kb, FIG. 52 b) allowed detection of thesite-specific integration of the gene cassette into the chloroplastgenome (9). FIG. 52 a shows the BglII sites used for the restrictiondigestion of the chloroplast DNA for pLD-VK1. The transformedchloroplast genome digested with BglII produced fragments of 5.2 kb and3.0 kb for pLDVK1 (FIG. 52 e), while the untransformed chloroplastgenome that had been digested with BglII formed a 4.4-kb fragment. Theflanking sequence probe can also show if homoplasmy of the chloroplastgenome had been achieved through the three rounds of selection. Theplants expressing PA showed slight degree of heteroplasmy in one or twotransgenic lines, as few of the wild-type genomes were not transformed.This is not uncommon and could be eliminated by germinating seeds onstringent selection medium containing 500 μg/ml spectinomycin. Thegene-specific probe with a size of approximately 0.52 kb was used toshow the specific gene integration producing a 3-kb fragment containingthe pagA gene as shown in FIG. 52 f.

Immunoblot Detection of PA Expression.

To determine whether the transgenic plants were producing PA, immunoblotanalysis was performed on leaf extracts. Probing blots with anti-PAmonoclonal antibody revealed full-length 83-kDa protein (FIG. 53 a). PAhas protease-sensitive sequences at residues 164 and 314 that are easilycleaved by trypsin and chymotrypsin, respectively, resulting inpolypeptides of 63 kDa and 20 kDa (for trypsin) or 47 kDa and 37 kDa(for chymotrypsin).

The absence of these or other such bands demonstrates that PA is intactwithin the chloroplast (FIG. 53 a). The supernatant samples fromwild-type plants did not show any band, indicating that anti-PAantibodies did not cross-react with any plant proteins in the crudeextract.

Quantification of PA using ELISA.

The PA protein expression levels of pLD-VK1 plants of T0 generationreached up to 4.5% of TSP in mature leaves under normal illuminationconditions (16 h of light and 8 h of dark, FIG. 53 b). The psbAregulatory sequences, including the promoters and UTRs, have been shownto enhance translation and accumulation of foreign proteins undercontinuous light (12). Therefore, the pLD-VK1 transgenic lines wereexposed to continuous light an expression patterns were determined ondays 1, 3, 5, and 7 (FIG. 53 b). PA expression levels reached a maximumof 14.2% of the TSP in mature leaves at the end of day 5 and theexpression levels declined to 11.7% TSP on day 7. The larger amount ofPA in mature leaves is probably due to the high number of chloroplastsin mature leaves and the high copy number of chloroplast genomes (up to10,000 copies per cell). The decrease in PA expression in bleached oldleaves could be due to degradation of the proteins during senescence.These results show that approximately 1.8 mg PA can be obtained per gramfresh weight of mature leaf upon exposure to 5-day continuousillumination. Thus, approximately 150 mg of PA can be obtained from asingle plant and with 8,000 tobacco plants on an acre of land, 1.2 kg ofPA can be obtained per single cutting of tobacco plant (petit Havanavariety, Table 1). Upon three cuttings in a year, a total of 3.6 kg ofPA can be obtained. Assuming a loss of 50% during purification and 5 μgPA per dose (current vaccine dose is in a range of 1.75 to 7 μg PA)(17), a total of 360 million doses of vaccine can be obtained per acreof land. The commercial cultivar yields 40 metric tons biomass of freshleaves as opposed to 2.2 tons in experimental cultivar petit Havana (7).Therefore, the commercial cultivar is expected to give 18-fold-higheryields than the experimental cultivar. Thus an acre of land grown withtransgenic tobacco plants would yield vaccine sufficient for a verylarge population.

Functional Analysis of PA with Macrophage Cytotoxicity Assay.

FIG. 54 a shows the Coomassie-stained gel of crude leaf extracts andvarious purification fractions and the absence of PA in the flowthrough.The expression level of PA is so high that it can be observed in aCoomassie-stained gel even in crude plant extracts. FIG. 54 b is aCoomassie-stained gel showing fractions of purified and concentratedchloroplast derived PA used for immunization studies. Supernatantsamples from crude extracts of plant leaves expressing PA and partiallypurified chloroplast-derived PA were tested for functionality in vitrousing the well-defined macrophage lysis assay (16). The transgenicplants were shown to produce fully functional PA (FIG. 55). Crudeextracts of wild-type tobacco plant and plant extraction buffer wereused as negative controls. The crude extract of plant leaves expressingPA had activity equal to that of a 20-μg/ml solution of purified B.anthracis-derived PA. These results show that the PA expressed in plantshas high functional activity.

Immunization of BALB/c Mice.

Having confirmed that the chloroplast-derived PA has in vitro biologicalactivity comparable to that of the B. anthracis-derived PA, we proceededfurther to investigate the functionality in vivo. For this, seven groupseach consisting of five mice were injected s.c. with 5 μg of the antigenon days 0, 14, 28, and 140. The group 1 and group 2 mice, immunized withchloroplast-derived partially purified PA and with B. anthracis-derivedfully purified PA, respectively, both adsorbed to alhydrogel adjuvant,showed comparable IgG immune titers of about 1:300,000 (FIG. 56 a).These observations are comparable to those of earlier studies whereanti-PA titers up to 1:250,000 were observed in guinea pigs immunizedwith PA along with adjuvant (4). The observation that thechloroplast-derived PA and PA derived from B. anthracis show comparableimmune responses suggests that the plant-derived PA has been properlyfolded and was fully functional. The group that received partiallypurified chloroplast-derived PA without adjuvant showed titers rangingfrom 1:10,000 to 1:40,000, while the mice that received PA derived fromB. anthracis without adjuvant showed titers of 1:80,000 to 1:160,000.Previous studies showed that mice immunized s.c. with recombinant PA(rPA) derived from B. anthracis along with the adjuvant had significantantibody titers, while no significant immune response was observed inthe group immunized with PA alone (13). Similarly, guinea pigs immunizeds.c. with rPA derived from B. subtilis did not elicit a significant IgGimmune response, while rPA with alhydrogel adjuvant showed significantlevels of IgG titers above 1:15,000 (20). Taken together, these studiesshow that PA alone may not be a potent immunogen to elicit a significantimmune response and therefore all currently used anthrax vaccinescontain an adjuvant.

The difference between the immune responses between the two groupsimmunized with chloroplast-derived PA and B. anthracis-derived PA couldbe due to differences in the purities of the proteins. The level ofpurity was extremely high in PA derived from B. anthracis because of theuse of anion-exchange and gel filtration chromatography and fast proteinliquid chromatography (FPLC) to eliminate the breakdown products of thePA (21), whereas chloroplast-derived PA was purified by affinitychromatography without using protease inhibitors. In the presence ofadjuvant, PA binds to the alhydrogel via electrostatic forces (21),making it more stable against proteolytic degradation. Differences inthe titer values of the groups that received PA with and withoutadjuvant were probably due to depot effect (2) and due to thealhydrogel's nonspecific priming of the immune system. The group thatreceived transgenic plant crude extracts expressing PA with adjuvantshowed IgG titers ranging from 1:40,000 to 1:80,000. In spite ofsignificant levels of impurities in the crude extract, this group showedgood immune titers, confirming high expression levels of PA intransgenic leaves.

Toxin Neutralization Assay of Serum Samples.

In order to evaluate the functionality of the IgG antibodies produced inresponse to the immunization, sera from the mice were tested for theirability to neutralize PA and thereby protect macrophages against LTkilling Toxin neutralization assays were performed on two different setsof sera. The first set was drawn 15 days after the third immunizationdose (day 43 of post initial immunization), and the second set was drawn15 days after the fourth immunization dose (day 155 of post-initialimmunization). Sera obtained after the third dose (FIG. 56 b) showedsimilar neutralization titers for the mice immunized withchloroplast-derived PA or B. anthracis-derived PA when both proteinswere administered with adjuvant (1:10,000 to 1:100,000). Theseobservations are in agreement with the results obtained in earlierstudies where neutralization titers of 20,000 to 70,000 were obtainedwhen guinea pigs were immunized with PA derived from B. anthracis alongwith adjuvant (4). However, titers were slightly higher for B.anthracis-derived PA used in conjunction with adjuvant in bleeds afterthe fourth immunization (FIG. 56 c). The mice immunized withchloroplast-derived PA alone showed significantly smaller neutralizationtiters (between 1:100 and 1:1,000) than the mice immunized with B.anthracis-derived PA alone (1:10,000 to 1:200,000 after the thirdimmunization and 1:10,000 to 1:50,000 after the fourth immunization,FIG. 56 b and c). Mice immunized with the crude extracts ofPA-expressing leaves showed strong neutralization titers, ranging from1:500 to 1:7500, with the exception of a single mouse after the fourthimmunization (FIG. 56 c). Control mice immunized with wild-type plantleaf crude extract or PBS did not show any immune response orneutralization ability. Generally, the average neutralization titerscompared among different groups showed similar distribution patterns tothat of the average anti-PA immune titers determined by the ELISA. Theseresults show that there is good correlation between the anti-PA antibodylevels and neutralization titers.

Toxin challenge of BALB/c mice. We proceeded to test the immunized micefor their ability to survive challenge with 1.5×100% lethal dose (LD100)of LT (22). Mice immunized with the chloroplast or B. anthracis-derivedPA with adjuvant survived the toxin challenge. Mice immunized with crudeextracts of plants expressing PA showed a significant survival rate of80%, confirming high PA expression levels. In this group, 4 out of 5mice showed neutralization titers above 1:1,000. These studiesdemonstrate the immunoprotective properties of chloroplast-derived PAagainst anthrax LT challenge. The single mouse in this group that showeda neutralization titer below 1:150 may have been the one to succumb.None of the mice immunized with chloroplast-derived PA without adjuvantsurvived (FIG. 57), as expected from their low neutralization titers(FIG. 56 b and c). The comparison of neutralization titers to mousechallenge survival for all the groups seems to indicate neutralizationtiters at and above 1:1,000 result in protection against challenge withgreater than LD100 doses of LT. These results prove the immunogenic andimmunoprotective properties of plant-derived B. anthracis PA. Priorstudies did not investigate functionality of plant-derived PA in animalstudies (3, 30). The production of anti-PA IgG antibodies combined within vitro neutralization and toxin challenge studies shows thatimmunization with transgenic chloroplast-derived PA is highly effective.Plant-derived recombinant PA is free of EF and LF and easy to produce,without the need for expensive fermenters. Even with 50% loss duringpurification, 1 acre of transgenic plants can produce 360 million dosesof functional anthrax vaccine. Our studies open the door for possibleoral immunization through feeding of edible plant parts like carrotroots, which should effectively stimulate the mucosal immune system aswell as a systemic immune response, thereby offering better protectionagainst pathogens that attack through mucosa. Delivering vaccines inedible plants can potentially eliminate existing vaccine purificationand processing steps, cold storage and transportation requirements, andthe need for health professionals for vaccine delivery. Although foreigngenes have been expressed in chromoplasts of edible plant parts (18),there is no report of expressing vaccine antigens in non-green plastidspresent within edible tissues so far. In addition to maternalinheritance of transgenes engineered via the chloroplast genomes (8),cytoplasmic male sterility has been developed as another fail-safemechanism for biological containment of transgenes (27). Furthermore,successful engineering of several foreign operons via the chloroplastgenome (24) has opened the door for development of multivalent vaccines.

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Finally, while various embodiments of the present invention have beenshown and described herein, it will be obvious that such embodiments areprovided by way of example only. Numerous variations, changes andsubstitutions may be made without departing from the invention herein.Accordingly, it is intended that the invention be limited only by thespirit and scope of the appended claims. The teachings of all patentsand other references cited herein are incorporated herein by referencein their entirety to the extent they are not inconsistent with theteachings herein.

1-78. (canceled)
 79. A method of retarding the development of diabetesin a subject in need thereof comprising obtaining a compositioncomprising a CTB-Pris polypeptide and at least one chloroplast genomehaving a transgene inserted therein that encodes said CTB-Prispolypeptide and administering to said subject said composition; whereinsaid CTB-Pris polypeptide comprises an insulin A-chain, an insulinB-chain and an insulin C-peptide; and wherein said CTB-Pris comprises afurin cleavage site located between CTB and proinsulin on the CTB-Prispolypeptide.
 80. The method of claim 79, wherein said CTB-Prispolypeptide comprises a furin cleavage site located between said insulinA-chain and said insulin C-peptide.
 81. The method of claim 79, whereinsaid CTB-Pris polypeptide comprises a furin cleavage site locatedbetween said insulin B-chain and said insulin C-peptide.
 82. A methodfor vaccinating a human against rotavirus infection, comprisingadministering to the human an immunizing amount of a compositioncomprising a CTB-NSP4 fusion protein, wherein said CTB-NSP4 fusionprotein is derived from a plant transformed to express said CTB-NSP4fusion protein.
 83. A method for vaccinating a human against Hepatitis Cinfection, comprising administering to the human an immunizing amount ofa composition comprising a Hepatitis C NS3 antigen polypeptide, whereinsaid Hepatitis C NS3 antigen polypeptide is derived from a planttransformed to express said Hepatitis C NS3 antigen polypeptide.